Method of synthesizing thyroid hormone analogs and polymorphs thereof

ABSTRACT

The disclosure describes methods of synthesis of pyridazinone compounds as thyroid hormone analogs and their prodrugs. Preferred methods according to the disclosure allow for large-scale preparation of pyridazinone compounds having high purity. In some embodiments, preferred methods according to the disclosure also allow for the preparation of pyridazinone compounds in better yield than previously used methods for preparing such compounds. Also disclosed are morphic forms of a pyridazinone compound. Further disclosed is a method for treating resistance to thyroid hormone in a subject having at least one TRβ mutation.

RELATED APPLICATIONS

This application is a continuation application of InternationalApplication PCT/US2013/060177 with an international filing date of Sep.17, 2013, which claims priority to, and the benefit of, U.S. provisionalapplication No. 61/702,137, filed Sep. 17, 2012 and U.S. provisionalapplication No. 61/790,432, filed Mar. 15, 2013, the entire contents ofeach of which are incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “41245-522001WO_ST25.txt”, which wascreated on Sep. 16, 2013 and is 4 KB in size, are hereby incorporated byreference in their entirety.

BACKGROUND

Thyroid hormones are critical for normal growth and development and formaintaining metabolic homeostasis (Paul M. Yen, Physiological reviews,Vol. 81(3): pp. 1097-1126 (2001)). Circulating levels of thyroidhormones are tightly regulated by feedback mechanisms in thehypothalamus/pituitary/thyroid (HPT) axis. Thyroid dysfunction leadingto hypothyroidism or hyperthyroidism clearly demonstrates that thyroidhormones exert profound effects on cardiac function, body weight,metabolism, metabolic rate, body temperature, cholesterol, bone, muscleand behavior.

The biological activity of thyroid hormones is mediated by thyroidhormone receptors (TRs or THRs) (M. A. Lazar, Endocrine Reviews, Vol.14: pp. 348-399 (1993)). TRs belong to the superfamily known as nuclearreceptors. TRs form heterodimers with the retinoid receptor that act asligand-inducible transcription factors. TRs have a ligand bindingdomain, a DNA binding domain, and an amino terminal domain, and regulategene expression through interactions with DNA response elements and withvarious nuclear co-activators and co-repressors. The thyroid hormonereceptors are derived from two separate genes, α and β. These distinctgene products produce multiple forms of their respective receptorsthrough differential RNA processing. The major thyroid receptor isoformsare α1, α2, β1 and β2. Thyroid hormone receptors α1, β1 and β2 bindthyroid hormone. It has been shown that the thyroid hormone receptorsubtypes can differ in their contribution to particular biologicalresponses. Recent studies suggest that TRβ1 plays an important role inregulating TRH (thyrotropin releasing hormone) and on regulating thyroidhormone actions in the liver. TRβ2 plays an important role in theregulation of TSH (thyroid stimulating hormone) (Abel et. al., J. Clin.Invest., Vol 104: pp. 291-300 (1999)). TRβ1 plays an important role inregulating heart rate (B. Gloss et. al. Endocrinology, Vol. 142: pp.544-550 (2001); C. Johansson et. al., Am. J. Physiol., Vol. 275: pp.R640-R646 (1998)).

Efforts have been made to synthesize thyroid hormone analogs whichexhibit increased thyroid hormone receptor beta selectivity and/ortissue selective action. Such thyroid hormone mimetics may yielddesirable reductions in body weight, lipids, cholesterol, andlipoproteins, with reduced impact on cardiovascular function or normalfunction of the hypothalamus/pituitary/thyroid axis (see, e.g.,Joharapurkar et al., J. Med. Chem., 2012, 55 (12), pp 5649-5675). Thedevelopment of thyroid hormone analogs which avoid the undesirableeffects of hyperthyroidism and hypothyroidism while maintaining thebeneficial effects of thyroid hormones would open new avenues oftreatment for patients with metabolic disease such as obesity,hyperlipidemia, hypercholesterolemia, diabetes and other disorders anddiseases such as liver steatosis and NASH, atherosclerosis,cardiovascular diseases, hypothyroidism, thyroid cancer, thyroiddiseases, resistance to thyroid hormone and related disorders anddiseases.

The present invention, in part, provides methods for synthesizingthyroid hormone analogs such as pyridazinone compounds and prodrugsthereof. An ideal method of synthesizing the thyroid hormone analogs andtheir prodrugs would, for example, provide product compounds in highpurity and high yield. The present invention is directed at providingone or more of these desirable features.

SUMMARY OF THE DISCLOSURE

The present disclosure describes a synthetic process, which may be usedto prepare6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (“Int.7”), a compound that is useful as an intermediate for makingpyridazinone compounds as thyroid hormone analogs, as follows:

(a) contacting R¹MgX or R¹Li with a compound of Formula (I):

to form a compound of Formula (II):

in which R¹ is isopropyl or isopropenyl, X is halo and R² is H or anamine protecting group; and

(b) converting the compound of Formula (II) to a compound of Formula(III):

-   -    in the presence of a base when R¹ is isopropenyl or in the        presence of an oxidizing agent when R¹ is isopropyl.

In step (a), the solvent can be an aprotic organic solvent, such as THF,diethyl ether, toluene, or dioxane, the reaction temperature can be0-60° C., 20-50° C., 30-45° C., or 35-45° C., the reaction time can be10 min to 10 hours, 1-8 hours, or 3-5 hours, and the amount of theGrignard reagent (R¹MgX) can be 3-10 equivalents or 3-6 equivalents ofthe compound of Formula (I).

In step (b), the base is used to isomerize the compound of Formula (II).It can be an organic base or an inorganic base. Examples of basesinclude, but are not limited to, triethylamine, pyridine, KOH, NaOH, andcarbonates. The isomerization can also be achieved under otherconditions, e.g., treatment with an acid or heating in an aproticsolvent.

Also, in step (b), the oxidizing agent is not particularly limited. Forexample, one can use bromine in acetic acid or propionic acid.

Examples of amine protecting groups include, but are not limited to,substituted alkyl, acyl (e.g., benzoyl or acetyl) and silyl. Hydroxy andamine protecting groups have been discussed in T. W. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley andSons (1991).

In one embodiment, step (a) is performed by contacting R¹MgX with thecompound of Formula (I), in which R¹ is isopropenyl and X is Br. Thesolvent used in this reaction can be THF with a volume to weight ratioof THF to the compound of Formula (I) ranging between 7 and 30 (orbetween 7 and 15). This step may be performed in the presence of a Lewisacid (e.g., a lithium halide).

In one embodiment, step (a) is performed by contacting R¹MgX with thecompound of Formula (I), in which R¹ is isopropyl and X is Cl. Thesolvent used in this reaction can be THF with a volume to weight ratioof THF to the compound of Formula (I) ranging between 7 and 30 (orbetween 7 and 15). This step may be performed in the presence of a Lewisacid (e.g., a lithium halide).

In one embodiment, the base in step (b) is a metal hydroxide (e.g.,potassium hydroxide).

In one embodiment, the oxidizing agent in step (b) is bromine and step(b) is performed in the presence of an acid.

In one embodiment, the R² group in Formula (I) and Formula (II) isacetyl or benzoyl. In a further embodiment, R² is benzoyl.

In one embodiment, the process further comprises providing the compoundof Formula (I) by contacting 3,6-dichloropyridazine with2,6-dichloro-4-aminophenol to form3,5-dichloro-4-((6-chloropyridazin-3-yl)oxy)aniline, hydrolyzing3,5-dichloro-4-((6-chloropyridazin-3-yl)oxy)aniline and protecting theamine group of 3,5-dichloro-4-((6-chloropyridazin-3-yl)oxy)anilineeither before or after the hydrolysis to form the compound of Formula(I). The contacting of 3,6-dichloropyridazine with2,6-dichloro-4-aminophenol is performed in a polar aprotic solvent(e.g., dimethylacetamide (DMAC)) in the presence of a base (e.g.,Cs₂CO₃) at a reaction temperature between 60 and 120° C. (e.g., about65° C.). Further, a purification step may be included. That is, beforestep (a), the compound of Formula (I) is purified in an acidic solutionat a temperature between 80 and 100° C.

In one embodiment, the process further comprises step (c) when present,removing the amine protecting group R² of the compound of Formula (III)to form 6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one.

In one embodiment, the compound, e.g., Int. 7, made by the methoddescribed herein has a purity of greater than 85%, e.g., greater than86%, greater than 90%, greater than 92.5%, greater than 95%, greaterthan 96%, greater than 97%, greater than 97.5%, greater than 98%,greater than 98.5%, greater than 99%, greater than 99.2%, greater than99.5%, or greater than 99.8%.

In one embodiment, the compound, i.e.,6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one, made bythe method described herein has less than 1.5% of6-(4-amino-2,6-dichlorophenoxy)-5-isopropylpyridazin-3(2H)-one, e.g.,less than 1.0% of6-(4-amino-2,6-dichlorophenoxy)-5-isopropylpyridazin-3(2H)-one, or lessthan 0.5% of6-(4-amino-2,6-dichlorophenoxy)-5-isopropylpyridazin-3(2H)-one.

In another embodiment, the compound made by the above-described processis free of6-(4-amino-2,6-dichlorophenoxy)-5-isopropylpyridazin-3(2H)-one.

The synthetic process of this invention may further comprise thefollowing step to synthesize pyridazinone compounds as thyroid hormoneanalogs and their prodrugs:

-   (d) converting    6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one to    the compound of Formula (IV):

wherein

R³ is H or CH₂R_(a), in which R_(a) is hydroxyl, O-linked amino acid,—OP(O)(OH)₂ or —OC(O)—R_(b), R_(b) being lower alkyl, alkoxy, alkylacid, cycloalkyl, aryl, heteroaryl, or —(CH₂)_(n)-heteroaryl and n being0 or 1;

R⁴ is H, and R⁵ is CH₂COOH, C(O)CO₂H, or an ester or amide thereof, orR⁴ and R⁵ together are —N═C(R_(c))—C(O)—NH—C(O)—; in which R_(c) is H orcyano.

In one embodiment, the compound of Formula (IV) is2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) and the above step is performed by contacting6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one withethyl (2-cyanoacetyl)carbamate and a metal nitrite followed by treatmentwith potassium acetate in DMAC.

In one embodiment, the process further comprises forming a morphic formof2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) (Form I) characterized by an X-ray powder diffractionpattern including peaks at about 10.5, 18.7, 22.9, 23.6, and 24.7degrees 2θ.

In one embodiment, the compound of Formula (IV) is of Formula (V)

-   -    wherein R³ is CH₂R_(a), and step (d) is performed by contacting        6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one        with ethyl (2-cyanoacetyl)carbamate followed by treatment with        potassium acetate in DMAC to form        2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile        (“Compound A”) and converting Compound A to the compound of        Formula (V) in a suitable manner, e.g., using one of the        techniques described in U.S. Pat. No. 8,076,334.

In one embodiment, the compound of Formula (IV), e.g.,2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”), made by the method described herein has a purity ofgreater than 85%, e.g., greater than 86%, greater than 90%, greater than92.5%, greater than 95%, greater than 96%, greater than 97%, greaterthan 97.5%, greater than 98%, greater than 98.5%, greater than 99%,greater than 99.2%, greater than 99.5%, or greater than 99.8%. Forexample, the content of impurities (i.e., any components of thecomposition produced by the method described herein, other than compoundof Formula (IV), such as byproducts, starting material, solventresidues, heavy metal, and etc.) is less than 15%, less than 14%, lessthan 10%, less than 8%, less than 5%, less than 4%, less than 3%, lessthan 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%,or less than 0.2%.

In one embodiment, the compound of Formula (IV) made by the methoddescribed herein is Compound A in Form I, and has a purity of greaterthan 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%,greater than 95%, greater than 96%, greater than 97%, greater than97.5%, greater than 98%, greater than 98.5%, greater than 99%, greaterthan 99.2%, greater than 99.5%, or greater than 99.8%. For example, thecontent of impurities (i.e., any components of the composition producedby the method described herein, other than Compound A, such asbyproducts, starting material, solvent residues, heavy metal, and etc.)is less than 15%, less than 14%, less than 10%, less than 8%, less than5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than1%, less than 0.8%, less than 0.5%, or less than 0.2%.

In one embodiment, the compound of Formula (IV) made by the methoddescribed herein is Compound A in Form I, and Form I has a purity ofgreater than 85%, e.g., greater than 86%, greater than 90%, greater than92.5%, greater than 95%, greater than 96%, greater than 97%, greaterthan 97.5%, greater than 98%, greater than 98.5%, greater than 99%,greater than 99.2%, greater than 99.5%, or greater than 99.8%. Forexample, the content of impurities (i.e., any components of thecomposition produced by the method described herein, other than Form I,such as other morphic forms of Compound A, byproducts, startingmaterial, solvent residues, heavy metal, and etc.) is less than 15%,less than 14%, less than 10%, less than 8%, less than 5%, less than 4%,less than 3%, less than 2%, less than 1.5%, less than 1%, less than0.8%, less than 0.5%, or less than 0.2%.

In one embodiment, the composition comprising a compound of Formula(IV), such as Compound A, made by the method described herein, has lessthan 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of thecorresponding β-isopropylpyridazin-3(2H)-one regioisomer (e.g.,2-(3,5-dichloro-4-(4-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile,the β-isopropylpyridazin-3(2H)-one regioisomer of Compound A).

In one embodiment, the composition comprising a compound of Formula(IV), such as Compound A, made by the method described herein is free ofthe corresponding β-isopropylpyridazin-3(2H)-one regioisomer (e.g.,2-(3,5-dichloro-4-(4-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile,the β-isopropylpyridazin-3(2H)-one regioisomer of Compound A).

In one embodiment, the composition comprising a compound of Formula(IV), such as Compound A, made by the method described herein has lessthan 1.5% (e.g., less than 0.1%) of heavy metal, e.g., silver.

In one embodiment, the composition comprising a compound of Formula(IV), such as Compound A, made by the method described herein is free ofheavy metal, e.g., silver, gold, or platinum.

The synthetic methods described herein include advantages compared tothe previous methods, such as those disclosed in U.S. Pat. No.7,452,882. For example, the overall yield of2-(3,5-dichloro-4-(5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) is greatly increased (e.g., >40% versus ˜9% when madeaccording to the method disclosed in U.S. Pat. No. 7,452,882). Also,regioselectivity of the synthesis is far superior. Further, the newmethods offer easier processing, e.g., easier filtrations. Lastly, noheavy metals are used in the methods described herein for Compound A. Incomparison, silver was used in the route described in U.S. Pat. No.7,452,882, which necessitated remediation treatment with a resin.

In yet another aspect, the invention features a composition comprisinggreater than 85% of a compound of Formula (IV), less than 1.5% of thecorresponding β-isopropylpyridazin-3(2H)-one regioisomer (i.e.,

and/or has less than 1.5% of heavy metal.

In one embodiment, the compound of Formula (IV), e.g., Compound A, has apurity of greater than 85%, e.g., greater than 86%, greater than 90%,greater than 92.5%, greater than 95%, greater than 96%, greater than97%, greater than 97.5%, greater than 98%, greater than 98.5%, greaterthan 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.For example, the content of impurities (i.e., any components of acomposition comprising the compound of Formula (IV), other than thecompound of Formula (IV), such as byproducts, starting material, solventresidues, heavy metal, and etc.) is less than 15%, less than 14%, lessthan 10%, less than 8%, less than 5%, less than 4%, less than 3%, lessthan 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%,or less than 0.2%.

In one embodiment, the compound of Formula (IV) is Compound A in Form I,and has a purity of greater than 85%, e.g., greater than 86%, greaterthan 90%, greater than 92.5%, greater than 95%, greater than 96%,greater than 97%, greater than 97.5%, greater than 98%, greater than98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, orgreater than 99.8%. For example, the content of impurities (i.e., anycomponents of a composition comprising Compound A, other than CompoundA, such as byproducts, starting material, solvent residues, heavy metal,and etc.) is less than 15%, less than 14%, less than 10%, less than 8%,less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%,less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.

In one embodiment, the compound of Formula (IV) is Compound A in Form I,and Form I has a purity of greater than 85%, e.g., greater than 86%,greater than 90%, greater than 92.5%, greater than 95%, greater than96%, greater than 97%, greater than 97.5%, greater than 98%, greaterthan 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, orgreater than 99.8%. For example, the content of impurities (i.e., anycomponents of a composition comprising Form I, other than Form I, suchas other morphic forms of Compound A, byproducts, starting material,solvent residues, heavy metal, and etc.) is less than 15%, less than14%, less than 10%, less than 8%, less than 5%, less than 4%, less than3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, lessthan 0.5%, or less than 0.2%.

In one embodiment, the compound of Formula (IV), such as Compound A, hasless than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of thecorresponding β-isopropylpyridazin-3(2H)-one regioisomer (e.g.,2-(3,5-dichloro-4-((4-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile,the β-isopropylpyridazin-3(2H)-one regioisomer of Compound A).

In one embodiment, the compound of Formula (IV), such as Compound A, isfree of the corresponding β-isopropylpyridazin-3(2H)-one regioisomer(e.g.,2-(3,5-dichloro-4-((4-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile,the β-isopropylpyridazin-3(2H)-one regioisomer of Compound A).

In one embodiment, the compound of Formula (IV), such as Compound A, hasless than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of heavymetal, e.g., silver, gold, or platinum.

In one embodiment, the compound of Formula (IV), such as Compound A,made by the method described herein is free of heavy metal, e.g.,silver.

Further, the invention features a morphic form of2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) (Form I) characterized by an X-ray powder diffraction(“XRPD”) pattern including peaks at about 10.5, 18.7, 22.9, 23.6, and24.7 degrees 2θ.

In one embodiment, Form I is characterized by an X-ray powderdiffraction pattern further including peaks at about 8.2, 11.2, 15.716.4, 17.7, 30.0, and 32.2 degrees 2θ.

In one embodiment, Form I is characterized by an X-ray powderdiffraction pattern including peaks at about 8.2, 10.5, 18.7, 22.9,23.6, and 24.7 degrees 2θ.

In one embodiment, Form I is characterized by an X-ray powderdiffraction pattern including peaks at about 8.2, 10.5, 11.2, 15.7 16.4,17.7, 18.7, 22.9, 23.6, and 24.7 degrees 2θ.

In one embodiment, Form I is characterized by an X-ray powderdiffraction pattern including peaks at about 8.2, 10.5, 11.2, 15.7 16.4,17.7, 18.7, 22.9, 23.6, 24.7, 30.0, and 32.2 degrees 2θ.

In another embedment, Form I is characterized by an X-ray powderdiffraction pattern substantially similar to that set forth in FIG. 1.

In another aspect, the present disclosure describes a process ofpreparing Form I. The process comprises mixing a sample containingCompound A (e.g., either crude or purified preparation of Compound A)with an organic solvent, such as alcohol (e.g., ethanol), ketone (e.g.,methyl isobutyl ketone, i.e., MIBK), or an aqueous solution includingalcohol or ketone. For example, the resulting mixture (e.g., a slurry orsuspension) containing the staring Compound A and the solvent is heatedat a first temperature, and then cooled to a second temperature that islower than the first temperature. Preferably, the organic solvent isethanol. The starting Compound A which goes into the form conversion canbe a solvate, such as a hydrate (e.g., a monohydrate or dihydrate), or asolvate of an organic solvent (for example dimethyl acetamide, ethanolor MIBK). Alternatively, the starting Compound A can be an ansolvate(e.g., an anhydrate).

In one embodiment, the process is performed by heating the Compound Awith the organic solvent to an elevated temperature (e.g., about 60-110°C. or about 80° C.) to form a slurry or suspension, followed by cooling(e.g., to a temperature about 0-60° C., about 40-60° C., about 45-55°C., or at about room temperature) to give Compound A Form I. Forexample, the organic solvent is ethanol and slurry containing Compound Acan be cooled to a temperature greater than about 40° C. to obtain FormI. For example, the organic solvent is MIBK, and slurry containingCompound A can be cooled to room temperature to obtain Form I.

In another embodiment, an ethanol suspension of Compound A is heated toan elevated temperature (e.g., about 80° C.) and then cooled to atemperature not lower than about 40° C. (e.g., about 45-55° C.),filtered (e.g., about 45-55° C.), washed with warmed (e.g., 45-55° C.)ethanol and dried at e.g., 45-55° C. to obtain Form I of Compound A thatis substantially free of any solvate of Compound A such as ethanolsolvate. For example, Form I of Compound A as prepared has ethanolsolvate content of <5% (e.g., <2%, <1%, <0.5%, or <0.1%).

In one embodiment, the process further comprises, after cooling themixture, filtering the mixture. The filtration step can be performed ata temperature between about 0° C. and about 60° C. (e.g., about 40-60°C., about 45-55° C., or at about room temperature) to obtain a filtercake.

In one embodiment, the process further comprises, after filtering themixture, rinsing the filter cake. The rinsing step can be performed at atemperature between about 0° C. and about 60° C. (e.g., about 40-60° C.,about 45-55° C., or at about room temperature) with an organic solvent(e.g., an alcohol such as ethanol) to obtain a rinsed filter cake.

In one embodiment, the process further comprises, after rinsing thefilter cake, drying the rinsed filter cake. The drying step can beperformed at a temperature between about 0° C. and about 60° C. (e.g.,about 40-60° C., about 45-55° C., or at about room temperature) toobtain Form I of Compound A.

In one embodiment, Form I has a purity of greater than 91%, e.g.,greater than 92.5%, greater than 95%, greater than 96%, greater than97%, or greater than 97.5%.

In one embodiment, Form I has a purity of greater than 98%, e.g.,greater than 98.5%, greater than 99%, greater than 99.2%, greater than99.5%, or greater than 99.8%.

In another aspect, the disclosure provides compounds such as

and a salt thereof, e.g., useful in synthesizing6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (“Int.7”).

The disclosure also provides a method for treating a resistance tothyroid hormone (RTH) in a subject in need thereof. The method comprisesadministering to a subject having at least one TRβ mutation atherapeutically effective amount of a compound of Formula (IV):

wherein

-   -   R³ is H or CH₂R_(a), in which R_(a) is hydroxyl, O-linked amino        acid, —OP(O)(OH)₂ or —OC(O)—R_(b), R_(b) being lower alkyl,        alkoxy, alkyl acid, cycloalkyl, aryl, heteroaryl, or        —(CH₂)_(n)-heteroaryl and n being 0 or 1;    -   R⁴ is H, and R⁵ is CH₂COOH, C(O)CO₂H, or an ester or amide        thereof, or R⁴ and R⁵ together are —N═C(R_(c))—C(O)—NH—C(O)—; in        which R_(c) is H or cyano.

Resistance to thyroid hormone (RTH) is a syndrome characterized by avariable tissue hyposensitivity to thyroid hormone and is primarilycaused by autosomal dominant mutations to THRβ. See Shi et al.,Biochemistry 2005, 44, 4612-4626.

In one embodiment, the compound used in the above method is2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”), e.g., Compound A in Form I.

In one embodiment, the subject to be treated by the above method hasobesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholicsteatohepatitis, fatty liver, bone disease, thyroid axis alteration,atherosclerosis, a cardiovascular disorder, tachycardia, hyperkineticbehavior, hypothyroidism, goiter, attention deficit hyperactivitydisorder, learning disabilities, mental retardation, hearing loss,delayed bone age, neurologic or psychiatric disease or thyroid cancer.

In one embodiment, the THRβ mutation is selected from the groupconsisting of a substitution of threonine (T) for the wild type residuealanine (A) at amino acid position 234 of SEQ ID NO: 1 (A234T); asubstitution of glutamine (Q) for the wild type residue arginine (R) atamino acid position 243 of SEQ ID NO: 1 (R243Q); a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 316 of SEQ ID NO: 1 (R316H); and a substitution of threonine(T) for the wild type residue alanine (A) at amino acid position 317 ofSEQ ID NO: 1 (A317T). In another embodiment, the compound used in themethod restores activity of mutant THRβ.

In one embodiment, the purity of compound of Formula (IV), such asCompound A, is obtained from reslurrying a crude compound from asuitable solvent described herein. In another embodiment, the compoundis not a solvate (e.g., a hydrate).

In one embodiment, the compound of Formula (IV), e.g., Compound A, has apurity of greater than 85%, e.g., greater than 86%, greater than 90%,greater than 92.5%, greater than 95%, greater than 96%, greater than97%, greater than 97.5%, greater than 98%, greater than 98.5%, greaterthan 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.

In one embodiment, the compound of Formula (IV) is Compound A in Form I,and has a purity of greater than 85%, e.g., greater than 86%, greaterthan 90%, greater than 92.5%, greater than 95%, greater than 96%,greater than 97%, greater than 97.5%, greater than 98%, greater than98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, orgreater than 99.8%.

In one embodiment, the compound of Formula (IV) is Compound A in Form I,and Form I has a purity of greater than 85%, e.g., greater than 86%,greater than 90%, greater than 92.5%, greater than 95%, greater than96%, greater than 97%, greater than 97.5%, greater than 98%, greaterthan 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, orgreater than 99.8%.

In one embodiment, the compound of Formula (IV), such as Compound A, hasless than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of thecorresponding β-isopropylpyridazin-3(2H)-one regioisomer (e.g.,2-(3,5-dichloro-4-((4-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile,the β-isopropylpyridazin-3(2H)-one regioisomer of Compound A).

In one embodiment, the compound of Formula (IV), such as Compound A, isfree of the corresponding β-isopropylpyridazin-3(2H)-one regioisomer(e.g.,2-(3,5-dichloro-4-((4-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile,the β-isopropylpyridazin-3(2H)-one regioisomer of Compound A).

In one embodiment, the compound of Formula (IV), such as Compound A, hasless than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of heavymetal, e.g., silver, gold, or platinum.

In one embodiment, the subject is a mammal. In another embodiment, thesubject is a human.

The disclosure further provides a method for determining aresponsiveness of a subject to the compound of Formula (IV) or apharmaceutically acceptable salt thereof, the method comprising:

(a) providing a sample from the subject; and

(b) detecting a mutation in a thyroid hormone receptor (“TR”), whereinthe presence of the mutation indicates the subject is responsive to thecompounds or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula (IV) is2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”).

In one embodiment, the TR is TRβ.

In one embodiment, the subject treated by the method of this inventionhas obesity, hyperlipidemia, hypercholesterolemia, diabetes,non-alcoholic steatohepatitis, fatty liver, bone disease, thyroid axisalteration, atherosclerosis, a cardiovascular disorder, tachycardia,hyperkinetic behavior, hypothyroidism, goiter, attention deficithyperactivity disorder, learning disabilities, mental retardation,hearing loss, delayed bone age, neurologic or psychiatric disease orthyroid cancer.

In one embodiment, a method for determining a responsiveness to thecompound of Formula (IV) can be used together with the method fortreating a resistance to thyroid hormone. That is, before the treatment,a subject is tested to determine the responsiveness to the compound.

Other features and advantages of the present invention are apparent fromdetailed description, examples, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffractogram (XRPD) of2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) Form I.

FIG. 2 is a differential scanning calorimetry (DSC) diagram of CompoundA Form I.

FIGS. 3A and 3B are MacPymol modeling images to show T3 and Compound Ain THRβ, respectively.

FIG. 4 is a MacPymol modeling image to show superimposed T3 and CompoundA in THRβ.

FIG. 5A is a MacPymol modeling image to show polar interactions betweenT3 and wild type THRβ, where T3 interacts with Arg320 very specifically.

FIG. 5B is a MacPymol modeling image to show polar interactions betweenCompound A and wild type THRβ, where Compound A interacts with Arg320and Arg316.

FIG. 6 is a MacPymol modeling image to show that mutations lead to manychanges in the polar region of the ligand binding domain (“LBD”).

FIG. 7A is a MacPymol modeling image to show interactions between T3 andTHRβ mutants: Ala234Thr, Arg243Gln, Arg316His, Ala317Thr.

FIG. 7B is a MacPymol modeling image to show interactions betweenCompound A and THRβ mutants: Ala234Thr, Arg243Gln, Arg316His, Ala317Thr;indicating that, compared to T3, the negatively charged heterocycle inCompound A accommodates mutations better.

FIGS. 8A and 8B are MacPymol modeling images of T3 and Compound A inArg316His mutant, respectively. T3-Arg320 interaction is likely weakerdue to rotation of Arg320 away from ligand in the mutant, while CompoundA maintains favorable interaction with Arg320 and is well positioned forthe CN group to form a pi-cation interaction with the mutated His316.

FIGS. 9A and 9B are MacPymol modeling images of Compound A in the WTTHRβ and mutant Arg316His, respectively.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a reactant”includes not only a single reactant but also a combination or mixture oftwo or more different reactant, reference to “a substituent” includes asingle substituent as well as two or more substituents, and the like.

As used herein, the phrases “for example,” “for instance,” “such as,” or“including” are meant to introduce examples that further clarify moregeneral subject matter. These examples are provided only as an aid forunderstanding the disclosure, and are not meant to be limiting in anyfashion. Furthermore as used herein, the terms “may,” “optional,”“optionally,” or “may optionally” mean that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally present” means that an object may ormay not be present, and, thus, the description includes instanceswherein the object is present and instances wherein the object is notpresent.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, the abbreviation “TR” or “THR” refers to thyroid hormonereceptor. TR nucleic acids and polypeptides from various species (e.g.,human, rat, chicken, etc.) have previously been described. See, e.g., R.L. Wagner et al. (2001), Molecular Endocrinology 15(3): 398-410; J. Sapet al. (1986), Nature 324:635-640; C. Weinberger et al. (1986), Nature324:641-646; and C. C. Tompson et al. (1986), Science 237:1610-1614;each of which is incorporated herein by reference in its entirety. Theamino acid sequence of human TRβ is provided, e.g., by Genbank AccessionNo. P10828.2, incorporated herein by reference.

Amino acid sequence of the ligand bindingdomain (residues 203-461) of human TRβ (SEQ ID NO: 1)ELQKSIGHKPEPTDEEWELIKTVTEAHVATNAQGSHWKQKRKFLPEDIGQAPIVNAPEGGKVDLEAFSHFTKIITPAITRVVDFAKKLPMFCELPCEDQIILLKGCCMEIMSLRAAVRYDPESETLTLNGEMAVTRGQLKNGGLGVVSDAIFDLGMSLSSFNLDDTEVALLQAVLLMSSDRPGLACVERIEKYQDSFLLAFEHYINYRKHHVTHFWPKLLMKVTDLRMIGACHASRFLHMKVECPTELFP PLFLEVFED

The residues at the 234, 243, 316, and 317 positions of human TRβ areunderlined in SEQ ID NO: 1. The portion of the human TRβ nucleotidesequence that encodes the above amino acid sequence is SEQ ID NO: 2. Thenucleotide sequence of human TRβ is provided, e.g., by Genbank AccessionNo. NM_(—)000461.4, incorporated herein by reference.

Nucleic acid sequence encoding the ligand binding domain of human TRβ(SEQ ID NO: 2) GAGCTGCAGAAGTCCATCGGGCACAAGCCAGAGCCCACAGACGAGGAATGGGAGCTCATCAAAACTGTCACCGAAGCCCATGTGGCGACCAACGCCCAAGGCAGCCACTGGAAGCAAAAACGGAAATTCCTGCCAGAAGACATTGGACAAGCACCAATAGTCAATGCCCCAGAAGGTGGAAAGGTTGACTTGGAAGCCTTCAGCCATTTTACAAAAATCATCACACCAGCAATTACCAGAGTGGTGGATTTTGCCAAAAAGTTGCCTATGTTTTGTGAGCTGCCATGTGAAGACCAGATCATCCTCCTCAAAGGCTGCTGCATGGAGATCATGTCCCTTCGCGCTGCTGTGCGCTATGACCCAGAAAGTGAGACTTTAACCTTGAATGGGGAAATGGCAGTGACACGGGGCCAGCTGAAAAATGGGGGTCTTGGGGTGGTGTCAGACGCCATCTTTGACCTGGGCATGTCTCTGTCTTCTTTCAACCTGGATGACACTGAAGTAGCCCTCCTTCAGGCCGTCCTGCTGATGTCTTCAGATCGCCCGGGGCTTGCCTGTGTTGAGAGAATAGAAAAGTACCAAGATAGTTTCCTGCTGGCCTTTGAACACTATATCAATTACCGAAAACACCACGTGACACACTTTTGGCCAAAACTCCTGATGAAGGTGACAGATCTGCGGATGATAGGAGCCTGCCATGCCAGCCGCTTCCTGCACATGAAGGTGGAATGCCCCACAGAACTCTTCCCCCCTTTGTTCTTGGAAGTGTTCGAGGATTAG

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used. The term “independentlyselected from” is used herein to indicate that the recited elements,e.g., R groups or the like, can be identical or different.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group typically although not necessarilycontaining 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, aswell as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.Generally, although not necessarily, alkyl groups herein may contain 1to about 18 carbon atoms, and such groups may contain 1 to about 12carbon atoms. The term “lower alkyl” intends an alkyl group of 1 to 6carbon atoms, for example, 1, 2, 3, 4, 5, or 6 carbon atoms.“Substituted alkyl” refers to alkyl substituted with one or moresubstituent groups, and the terms “heteroatom-containing alkyl” and“heteroalkyl” refer to an alkyl substituent in which at least one carbonatom is replaced with a heteroatom, as described in further detailinfra.

The term “alkenyl” as used herein refers to a linear, branched or cyclichydrocarbon group of 2 to about 24 carbon atoms containing at least onedouble bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl,isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl,tetracosenyl, and the like. Generally, although again not necessarily,alkenyl groups herein may contain 2 to about 18 carbon atoms, and forexample may contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms. The term “substitutedalkenyl” refers to alkenyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkenyl” and“heteroalkenyl” refer to alkenyl in which at least one carbon atom isreplaced with a heteroatom, e.g., N, P, O, or S.

The term “alkynyl” as used herein refers to a linear or branchedhydrocarbon group of 2 to 24 carbon atoms containing at least one triplebond, such as ethynyl, n-propynyl, and the like. Generally, althoughagain not necessarily, alkynyl groups herein may contain 2 to about 18carbon atoms, and such groups may further contain 2 to 12 carbon atoms.The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbonatoms. The term “substituted alkynyl” refers to alkynyl substituted withone or more substituent groups, and the terms “heteroatom-containingalkynyl” and “heteroalkynyl” refer to alkynyl in which at least onecarbon atom is replaced with a heteroatom.

The term “alkoxy” as used herein intends an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group may berepresented as —O-alkyl where alkyl is as defined above. A “loweralkoxy” group intends an alkoxy group containing 1 to 6 carbon atoms,and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy,t-butyloxy, etc. Substituents identified as “C₁-C₆ alkoxy” or “loweralkoxy” herein may, for example, may contain 1 to 3 carbon atoms, and asa further example, such substituents may contain 1 or 2 carbon atoms(i.e., methoxy and ethoxy).

The term “alkyl acid” refers to an acid substituent that is on an alkylgroup, such as —(CH₂)_(o)COOH, in which o is an integer between 1 and 6.The alkyl group can either be linear or branched.

The term “aryl” as used herein, and unless otherwise specified, refersto an aromatic substituent generally, although not necessarily,containing 5 to 30 carbon atoms and containing a single aromatic ring ormultiple aromatic rings that are fused together, directly linked, orindirectly linked (such that the different aromatic rings are bound to acommon group such as a methylene or ethylene moiety). Aryl groups may,for example, contain 5 to 20 carbon atoms, and as a further example,aryl groups may contain 5 to 12 carbon atoms. For example, aryl groupsmay contain one aromatic ring or two fused or linked aromatic rings,e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine,benzophenone, and the like. “Substituted aryl” refers to an aryl moietysubstituted with one or more substituent groups, and the terms“heteroatom-containing aryl” and “heteroaryl” refer to aryl substituent,in which at least one carbon atom is replaced with a heteroatom, as willbe described in further detail infra. If not otherwise indicated, theterm “aryl” includes rings that are unsubstituted, substituted, and/orhave heteroatom-containing aromatic substituents.

The term “aralkyl” refers to an alkyl group with an aryl substituent,and the term “alkaryl” refers to an aryl group with an alkylsubstituent, wherein “alkyl” and “aryl” are as defined above. Ingeneral, aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms.Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbonatoms, and as a further example, such groups may contain 6 to 12 carbonatoms.

The term “amino” is used herein to refer to the group —NZ¹Z² wherein Z¹and Z² are hydrogen or nonhydrogen substituents, with nonhydrogensubstituents including, for example, alkyl, aryl, alkenyl, aralkyl, andsubstituted and/or heteroatom-containing variants thereof.

The terms “halo” and “halogen” are used in the conventional sense torefer to a chloro, bromo, fluoro or iodo substituent.

The term “heteroatom-containing” as in a “heteroatom-containing alkylgroup” (also termed a “heteroalkyl” group) or a “heteroatom-containingaryl group” (also termed a “heteroaryl” group) refers to a molecule,linkage or substituent in which one or more carbon atoms are replacedwith an atom other than carbon, e.g., nitrogen, oxygen, sulfur,phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly,the term “heteroalkyl” refers to an alkyl substituent that isheteroatom-containing, the term “heterocycle” or “heterocyclic” refersto a cyclic moiety that is heteroatom-containing, the terms “heteroaryl”and “heteroaromatic” respectively refer to “aryl” and “aromatic”substituents that are heteroatom-containing, and the like. Examples ofheteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl,N-alkylated amino alkyl, and the like. Examples of heteroarylsubstituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl,indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl,etc., and examples of heteroatom-containing alicyclic groups arepyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, etc.

“Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 toabout 30 carbon atoms, including 1 to about 24 carbon atoms, furtherincluding 1 to about 18 carbon atoms, and further including about 1 to12 carbon atoms, including linear, branched, cyclic, saturated andunsaturated species, such as alkyl groups, alkenyl groups, aryl groups,and the like. “Substituted hydrocarbyl” refers to hydrocarbylsubstituted with one or more substituent groups, and the term“heteroatom-containing hydrocarbyl” refers to hydrocarbyl in which atleast one carbon atom is replaced with a heteroatom.

The term “O-linked amino acid” means any amino acid, naturally occurringor synthetic, linked to a molecule via an oxygen of a carboxyl group ofthe amino acid, preferably via the carboxyl group of the carboxyterminus of the amino acid.

As used herein, the term “protecting group” means that a particularfunctional moiety, e.g., O, S, or N, is temporarily blocked so that areaction can be carried out selectively at another reactive site in amultifunctional compound. In preferred embodiments, a protecting groupreacts selectively in good yield to give a protected substrate that isstable to the projected reactions; the protecting group must beselectively removed in good yield by readily available, preferablynontoxic reagents that do not attack the other functional groups; theprotecting group forms an easily separable derivative (more preferablywithout the generation of new stereogenic centers); and the protectinggroup has a minimum of additional functionality to avoid further sitesof reaction. As detailed herein, oxygen, sulfur, nitrogen and carbonprotecting groups may be utilized. For example, in certain embodiments,certain exemplary oxygen protecting groups may be utilized. These oxygenprotecting groups include, but are not limited to methyl ethers,substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM(methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM(p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substitutedbenzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES(triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS(t-butyldimethylsilyl ether), tribenzyl silyl ether, and TBDPS(t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate(Bz), trifluoroacetate, and dichloroacetate), carbonates, cyclic acetalsand ketals. In certain other exemplary embodiments, nitrogen protectinggroups are utilized. Nitrogen protecting groups, as well as protectionand deprotection methods are known in the art. Nitrogen protectinggroups include, but are not limited to, carbamates (including methyl,ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclicimide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, andenamine derivatives. In yet other embodiments, certain exemplary sulfurprotecting groups may be utilized. The sulfur protecting groups include,but are not limited to those oxygen protecting group describe above aswell as aliphatic carboxylic acid (e.g., acrylic acid), maleimide, vinylsulfonyl, and optionally substituted maleic acid. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the present invention. Additionally, a variety of protectinggroups are described in “Protective Groups in Organic Synthesis” ThirdEd. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York:1999, the entire contents of which are hereby incorporated by reference.

By “substituted” as in “substituted hydrocarbyl,” “substituted alkyl,”“substituted aryl,” and the like, as alluded to in some of theaforementioned definitions, is meant that in the hydrocarbyl, alkyl,aryl, or other moiety, at least one hydrogen atom bound to a carbon (orother) atom is replaced with one or more non-hydrogen substituents.Examples of such substituents include, without limitation, functionalgroups and the hydrocarbyl moieties C₁-C₂₄ alkyl (including C₁-C₁₈alkyl, further including C₁-C₁₂ alkyl, and further including C₁-C₆alkyl), C₂-C₂₄ alkenyl (including C₂-C₁₈ alkenyl, further includingC₂-C₁₂ alkenyl, and further including C₂-C₆ alkenyl), C₂-C₂₄ alkynyl(including C₂-C₁₈ alkynyl, further including C₂-C₁₂ alkynyl, and furtherincluding C₂-C₆ alkynyl), C₅-C₃₀ aryl (including C₅-C₂₀ aryl, andfurther including C₅-C₁₂ aryl), and C₆-C₃₀ aralkyl (including C₆-C₂₀aralkyl, and further including C₆-C₁₂ aralkyl).

By “functional group,” as alluded to in some of the aforementioneddefinitions, is meant a non-hydrogen group comprising one or morenon-hydrocarbon functionality. Examples of functional groups include,without limitation: halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy(—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl(—(CO)—O-aryl), halocarbonyl (—CO)—X where X is halo), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂),mono-substituted C₁-C₂₄ alkylcarbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-substituted alkylcarbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂), mono-substitutedarylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH₂), carbamido(—NH—(CO)—NH₂), cyano (—C≡N), isocyano (—N⁺≡C⁻), cyanato (—O—C≡N),isocyanato (—O—N⁺≡C⁻), isothiocyanato (—S—C≡N), azido (—N═N⁺═N⁻), formyl(—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono- and di-(C₁-C₂₄alkyl)-substituted amino, mono- and di-(C₅-C₂₀ aryl)-substituted amino,C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₅-C₂₀ arylamido (—NH—(CO)-aryl),imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₀alkaryl, C₆-C₂₀ aralkyl, etc.), alkylimino (—CR═N(alkyl), whereR=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (—CR═N(aryl), whereR=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO),sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl;also termed “alkylthio”), arylsulfanyl (—S-aryl; also termed“arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl(—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl(—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂),phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino (—PH₂), mono- anddi-(C₁-C₂₄ alkyl)-substituted phosphino, mono- and di-(C₅-C₂₀aryl)-substituted phosphino; and the hydrocarbyl moieties C₁-C₂₄ alkyl(including C₁-C₁₈ alkyl, further including C₁-C₁₂ alkyl, and furtherincluding C₁-C₆ alkyl), C₂-C₂₄ alkenyl (including C₂-C₁₈ alkenyl,further including C₂-C₁₂ alkenyl, and further including C₂-C₆ alkenyl),C₂-C₂₄ alkynyl (including C₂-C₁₈ alkynyl, further including C₂-C₁₂alkynyl, and further including C₂-C₆ alkynyl), C₅-C₃₀ aryl (includingC₅-C₂₀ aryl, and further including C₅-C₁₂ aryl), and C₆-C₃₀ aralkyl(including C₆-C₂₀ aralkyl, and further including C₆-C₁₂ aralkyl). Inaddition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

The term “telescoping a process” refers to collapsing a multistepprocess into a smaller number of steps or unit operations. A unitoperation includes transformations, but also encompasses handling andisolation steps. Centrifugation, filtration, distillation, decantation,precipitation/crystallization, and packaging are examples of unitoperations. There are a great many examples of telescoping and otherprocess improvements in the literature (see, e.g., J. Org. Chem., 2007,72, 9757-9760).

It will be appreciated that some of the abovementioned definitions mayoverlap, such that some chemical moieties may fall within more than onedefinition.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl and aryl” isto be interpreted as “substituted alkyl and substituted aryl.”

The disclosure provides methods of synthesizing a compound, e.g., onethat is useful as an intermediate for synthesizing the pyridazinonecompounds as thyroid hormone analogs. Pyridazinone compounds as thyroidhormone analogs, as well as their prodrugs, have been disclosed in e.g.,U.S. Pat. Nos. 7,452,882, 7,807,674, and 8,076,334.

In particular, the invention features a method of making6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (“Int.7”) or a salt thereof, the method comprising:

-   -   (a) contacting R¹MgX or R¹Li with a compound of Formula (I):

to form a compound of Formula (II):

in which R¹ is isopropyl or isopropenyl, X is halo and R² is H or anamine protecting group; and

-   -   (b) converting the compound of Formula (II) to a compound of        Formula (III):

-   -    in the presence of a base when R¹ is isopropenyl or in the        presence of an oxidizing agent when R¹ is isopropyl.

The present disclosure also describes a method for synthesizing thepyridazinone compounds as thyroid hormone analogs, as well as theirprodrugs. Such compounds include those disclosed in U.S. Pat. Nos.7,452,882, 7,807,674, and 8,076,334. In particular, the disclosuredescribes a method of making a compound of Formula (IV) or apharmaceutically acceptable salt thereof:

wherein

-   -   R³ is H or CH₂R_(a), in which R_(a) is hydroxyl, O-linked amino        acid, —OP(O)(OH)₂ or —OC(O)—R_(b), R_(b) being lower alkyl,        alkoxy, alkyl acid, cycloalkyl, aryl, heteroaryl, or        —(CH₂)_(n)-heteroaryl and n being 0 or 1;    -   R⁴ is H, and R⁵ is CH₂COOH, C(O)CO₂H, or an ester or amide        thereof, or R⁴ and R⁵ together are —N═C(R_(c))—C(O)—NH—C(O)—; in        which R_(c) is H or cyano. The method comprises:

-   (a) contacting R¹MgX or R¹Li with a compound of Formula (I):

to form a compound of Formula (II):

in which R¹ is isopropyl or isopropenyl, X is halo and R² is H or anamine protecting group; and

-   -   (b) converting the compound of Formula (II) to a compound of        Formula (III):

-   -    in the presence of a base when R¹ is isopropenyl or in the        presence of bromine and an acid when R¹ is isopropyl,    -   (c) when present, removing the amine protecting group R² of the        compound of Formula (III) to form        6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one;        and, optionally    -   (d) converting        6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one        to the compound of Formula (IV) under a suitable condition.

The present invention also provides detailed methods for the synthesisof various disclosed compounds of the present invention according to thefollowing schemes and as shown in the Examples.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

In embodiments,6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (“Int.7”) is prepared according to Scheme 1 or 2 below.

Scheme 1: Synthesis of6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (Int. 7)with isopropyl Grignard reagent (iPrMgX)

Scheme 2: Synthesis of6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (Int. 7)with isopropenyl Grignard reagent

Stage 1: Synthesis of3,5-dichloro-4-((6-chloropyridazin-3-yl)oxy)aniline (Compound 2) andN-(3,5-dichloro-4-((6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)benzamideorN-(3,5-dichloro-4-((6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)acetamide(Compound 4)

Compound 2 is prepared by contacting 3,6-dichloropyridazine with2,6-dichloro-4-aminophenol in the presence of a small amount of asuitable base such as a metal carbonate (e.g., cesium or potassiumcarbonate) or a metal alkoxide (e.g., potassium t-butoxide) in asuitable organic solvent (e.g., DMSO or DMAC) at a suitable reactiontemperature (e.g., 60 to 120° C.) until completion of reaction,typically about 3 to 30 hours, for example about 3 to 15 hours.

Compound 4 is prepared by protecting 2 with a suitable amine protectingreagent (such as benzoic anhydride or benzoic chloride) followed bytreatment of the protected intermediate with sodium acetate in thepresence of a suitable organic solvent (such as acetic acid) at asuitable reaction temperature (e.g., 100 to 120° C.) until completion ofreaction, typically about 2 to 20 hours, for example about 5 to 15hours. The crude product is purified with a suitable solvent (e.g., amixture of water and acetic acid) at a suitable temperature (e.g.,88-100° C.). The acetate protected Compound 4 can be prepared bysubjecting Compound 2 to the hydrolysis conditions.

Stage 2: Synthesis ofN-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)benzamideorN-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)acetamide(Compound 6) and6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (Int. 7)

Compound 6 is prepared by contacting Compound 4 with an isopropylGrignard in a suitable organic solvent (such as tetrahydrofuran ordioxane) followed by an oxidation step. The oxidation step can beperformed in the presence of an oxidizing reagent such as bromine in asuitable organic solvent such as acetic acid at a suitable reactiontemperature (e.g., 60 to 90° C.) until completion of reaction, typicallyabout 2 to 10 hours, for example about 2 to 5 hours.

It will be appreciated that a deprotection reaction is required in orderto complete the transformation from Compound 6 to Int. 7. In particular,the N-protecting group (i.e., acetyl or benzoyl) must be removed inorder to obtain the free amino present in Int. 7. Thus, in oneembodiment, Int. 7 is obtained by deprotecting Compound 6 (where R² isBz) with a base such as metal hydroxide (e.g., KOH or NaOH) or metalcarbonate (e.g., sodium carbonate). In another embodiment, Int. 7 isobtained by deprotecting Compound 6 (where R² is Ac) with an acid suchas trifluoroacetic acid.

Alternatively, Compound 7 is prepared by contacting Compound 4 with anisopropenyl Grignard in a suitable organic solvent (such astetrahydrofuran or 2-methyl THF) followed by isomerization (e.g., from5A to 6) and deprotection under the treatment of a base such as metalhydroxide (e.g., KOH). The isomerization/deprotection step is performedat a suitable reaction temperature (e.g., 60 to 90° C.) until completionof reaction, typically about 10 to 60 hours, for example about 16 hoursat 90° C.

The Grignard reaction can be performed in the presence of a Lewis acidsuch as LiCl or LiBr at a suitable reaction temperature (e.g., roomtemperature to 40° C.) until completion of reaction, typically about 2to 10 hours, for example about 2 to 5 hours.

In embodiments, the synthesis of compound 5 or 5A results in improvedyield of Int. 7 relative to other methods known in the art. For example,the synthesis of 5 or 5A results in a yield of greater than 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85% or greater than 90%.

In embodiments, the Grignard reaction improves regioselectivity,resulting in significantly less β-isopropyl regioisomer of compound 6,i.e.,

and thus more pure Int. 7.

In one embodiment, the conversion from6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (“Int.7”) to Compound A is performed according to Scheme 3 below.

Stage 3: Synthesis of6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (Int. 8)

Int. 8 is prepared by contacting6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one withethyl (2-cyanoacetyl)carbamate and a metal nitrite such as sodiumnitrite in the presence of an acid (such as HCl) in a suitable solvent(e.g., a mixture of acetic acid and water) at a suitable reactiontemperature (e.g., below 10° C.) until the reaction is complete.

Stage 4: Synthesis of2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(Compound A)

Compound A is prepared by contacting Int. 8 and a base such as sodiumacetate or potassium acetate in a suitable solvent (e.g., DMAC) at asuitable reaction temperature (e.g., at about 120° C.) until thereaction is complete.

In embodiments, the conversion from6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (“Int.7”) to a compound of Formula (IV) other than MGL-3916 (such as prodrugsthereof) is performed under conditions described in, e.g., U.S. Pat.Nos. 7,452,882, 7,807,674, and 8,076,334, which are hereby incorporatedby reference in their entireties.

The synthetic methods described herein result in superiorregioselectivity, with the Grignard installation of the isopropenyl orisopropyl group versus the biaryl ether formation in the synthetic routepreviously disclosed in, e.g., U.S. Pat. No. 7,452,882, which gave poorregioselectivity. Further, by telescoping the biaryl ether formationinto the benzamide protection, the methods disclosed herein avoid theisolation of the biaryl ether product, which was nearly practicallyimpossible because of filtration times of greater than 1 week per batchwhen synthesizing this product in kilogram quantities.

The present invention provides, compounds with high purity and/or inspecific morphic form (e.g., Form I), compositions described herein andmethods for the treatment or prevention of obesity, hyperlipidemia,hypercholesterolemia, diabetes, non-alcoholic steatohepatitis, fattyliver, bone disease, thyroid axis alteration, atherosclerosis, acardiovascular disorder, tachycardia, hyperkinetic behavior,hypothyroidism, goiter, attention deficit hyperactivity disorder,learning disabilities, mental retardation, hearing loss, delayed boneage, neurologic or psychiatric disease or thyroid cancer.

It will be appreciated that the methods disclosed herein are suitablefor both large-scale and small-scale preparations of the desiredcompounds. In preferred embodiments of the methods described herein, thethyroid hormone analogs may be prepared on a large scale, for example onan industrial production scale rather than on an experimental/laboratoryscale. For example, a batch-type process according to the methods of thedisclosure allows the preparation of batches of at least 1 g, or atleast 5 g, or at least 10 g, or at least 100 g, or at least 1 kg, or atleast 100 kg of thyroid hormone analogs. Furthermore, the methods allowthe preparation of a thyroid hormone analog having a purity of at least98%, or at least 98.5% as measured by HPLC.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising a compound of Formula IV in combination with at least onepharmaceutically acceptable excipient or carrier.

A “pharmaceutical composition” is a formulation containing a compound ofthe present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient or carrier” means an excipient orcarrier that is useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the specification and claims includes both one andmore than one such excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is a metabolic disorder.

In the practice of the method of the present invention, an effectiveamount of any one of the compounds of this invention or a combination ofany of the compounds of this invention or a pharmaceutically acceptablesalt or ester thereof, is administered via any of the usual andacceptable methods known in the art, either singly or in combination.The compounds or compositions can thus be administered orally (e.g.,buccal cavity), sublingually, parenterally (e.g., intramuscularly,intravenously, or subcutaneously), rectally (e.g., by suppositories orwashings), transdermally (e.g., skin electroporation) or by inhalation(e.g., by aerosol), and in the form or solid, liquid or gaseous dosages,including tablets and suspensions. The administration can be conductedin a single unit dosage form with continuous therapy or in a single dosetherapy ad libitum. The therapeutic composition can also be in the formof an oil emulsion or dispersion in conjunction with a lipophilic saltsuch as pamoic acid, or in the form of a biodegradable sustained-releasecomposition for subcutaneous or intramuscular administration.

Useful pharmaceutical carriers for the preparation of the compositionshereof, can be solids, liquids or gases; thus, the compositions can takethe form of tablets, pills, capsules, suppositories, powders,enterically coated or other protected formulations (e.g. binding onion-exchange resins or packaging in lipid-protein vesicles), sustainedrelease formulations, solutions, suspensions, elixirs, aerosols, and thelike. The carrier can be selected from the various oils including thoseof petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,soybean oil, mineral oil, sesame oil, and the like. Water, saline,aqueous dextrose, and glycols are preferred liquid carriers,particularly (when isotonic with the blood) for injectable solutions.For example, formulations for intravenous administration comprisesterile aqueous solutions of the active ingredient(s) which are preparedby dissolving solid active ingredient(s) in water to produce an aqueoussolution, and rendering the solution sterile. Suitable pharmaceuticalexcipients include starch, cellulose, talc, glucose, lactose, talc,gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodiumstearate, glycerol monostearate, sodium chloride, dried skim milk,glycerol, propylene glycol, water, ethanol, and the like. Thecompositions may be subjected to conventional pharmaceutical additivessuch as preservatives, stabilizing agents, wetting or emulsifyingagents, salts for adjusting osmotic pressure, buffers and the like.Suitable pharmaceutical carriers and their formulation are described inRemington's Pharmaceutical Sciences by E. W. Martin. Such compositionswill, in any event, contain an effective amount of the active compoundtogether with a suitable carrier so as to prepare the proper dosage formfor proper administration to the recipient.

The pharmaceutical preparations can also contain preserving agents,solubilizing agents, stabilizing agents, wetting agents, emulsifyingagents, sweetening agents, coloring agents, flavoring agents, salts forvarying the osmotic pressure, buffers, coating agents or antioxidants.They can also contain other therapeutically valuable substances,including additional active ingredients other than those of formula I.

The compounds of the present invention are useful as medicaments for thetreatment of a resistance to thyroid hormone (RTH) in a subject who hasat least one TRβ mutation. The subject may have a disease, such asobesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholicsteatohepatitis, fatty liver, bone disease, thyroid axis alteration,atherosclerosis, a cardiovascular disorder, tachycardia, hyperkineticbehavior, hypothyroidism, goiter, attention deficit hyperactivitydisorder, learning disabilities, mental retardation, hearing loss,delayed bone age, neurologic or psychiatric disease or thyroid cancer.

The therapeutically effective amount or dosage of a compound accordingto this invention can vary within wide limits and may be determined in amanner known in the art. For example, the drug can be dosed according tobody weight. Such dosage will be adjusted to the individual requirementsin each particular case including the specific compound(s) beingadministered, the route of administration, the condition being treated,as well as the patient being treated. In another embodiment, the drugcan be administered by fixed does, e.g., dose not adjusted according tobody weight. In general, in the case of oral or parenteraladministration to adult humans, a daily dosage of from about 0.5 mg toabout 1000 mg should be appropriate, although the upper limit may beexceeded when indicated. The dosage is preferably from about 5 mg toabout 400 mg per day. A preferred dosage may be from about 20 mg toabout 100 mg per day. The daily dosage can be administered as a singledose or in divided doses, or for parenteral administration it may begiven as continuous infusion.

An effective amount of a pharmaceutical agent is that which provides anobjectively identifiable improvement as noted by the clinician or otherqualified observer. As used herein, the term “dosage effective manner”refers to amount of an active compound to produce the desired biologicaleffect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine,imidazole, lysine, arginine, morpholine, 2-hydroxyethylmorpholine,dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine,benzylamine, tetramethylammonium hydroxide and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present invention can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., an acetate, propionate or other ester.

The compounds of the present invention can also be prepared as prodrugs,for example, pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs in thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy or carbonyl group is bonded to any group that may becleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, New York-Oxford (1985).

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

The invention features a method for treating or alleviating a symptom ofresistance to thyroid hormone in a subject by administering to a subjectexpressing a mutant TRβ comprising a mutation in the ligand-bindingdomain a therapeutically effective amount of a compound of Formula (IV),such as Compound A, e.g., Form I thereof.

The disclosure also provides a method of determining a responsiveness ofa subject having resistance to thyroid hormone (RTH) to a compound ofFormula (IV) disclosed herein by providing a sample from the subject;and detecting at least one TRβ mutation (e.g., a gene mutation or amutation in the ligand-binding domain of TRβ polypeptide, e.g., apolypeptide as defined in SEQ ID NO: 1); and the presence of saidmutation indicates the subject is responsive to a compound of Formula(IV), such as Compound A, e.g., Form I thereof. The method can furtherinclude treating the subject who has the mutation by administering witha therapeutically effective amount of a compound of Formula (IV), suchas Compound A, e.g., Form I thereof.

In one embodiment, the subject that shows or will show responsiveness toa compound of Formula (IV) such as Compound A has obesity,hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholicsteatohepatitis, fatty liver, bone disease, thyroid axis alteration,atherosclerosis, a cardiovascular disorder, tachycardia, hyperkineticbehavior, hypothyroidism, goiter, attention deficit hyperactivitydisorder, learning disabilities, mental retardation, hearing loss,delayed bone age, neurologic or psychiatric disease or thyroid cancer.

Further, the disclosure also provides a method which includesdetermining the presence of a TRβ gene mutation in a sample from asubject; and selecting, based on the presence of an TRβ gene mutation, atherapy that includes the administration of a therapeutically effectiveamount of a compound of Formula (IV), such as Compound A, e.g., Form Ithereof.

The disclosure also provides a method which includes amplifying anucleic acid in a sample from a subject with a primer that iscomplementary to a mutant TRβ nucleic acid sequence comprising a TRβgene mutation in a nucleic acid sequence as defined in SEQ ID NO: 2;determining the presence of the amplified nucleic acid, and selecting,based on the presence of the amplified nucleic acid, a therapy thatincludes the administration of a therapeutically effective amount of acompound of Formula (IV), or treating the subject by administering atherapeutically effective amount of a compound of Formula (IV) based onthe presence of the amplified nucleic acid.

The mutant TRβ described herein is a mutant TRβ polypeptide or a nucleicacid sequence encoding a mutant TRβ polypeptide.

In one embodiment, the mutant TRβ comprises one or more mutations atamino acid positions 234, 243, 316, and 317 of SEQ ID NO: 1. Morepreferably, mutation is selected from the group consisting of asubstitution of threonine (T) for the wild type residue alanine (A) atamino acid position 234 of SEQ ID NO: 1 (A234T); a substitution ofglutamine (Q) for the wild type residue arginine (R) at amino acidposition 243 of SEQ ID NO: 1 (R243Q); a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 316 of SEQID NO: 1 (R316H); and a substitution of threonine (T) for the wild typeresidue alanine (A) at amino acid position 317 of SEQ ID NO: 1 (A317T).

In one embodiment, the mutant TRβ comprises a nucleic acid sequenceencoding a mutant TRβ polypeptide having one or more mutations at aminoacid positions 234, 243, 316, and 317 of SEQ ID NO: 1. A nucleic acidsequence encoding a mutant TRβ polypeptide or a peptide fragment that ischaracteristic of the mutant TRβ polypeptide can be detected using anysuitable method. For example, a nucleic acid sequence encoding a mutantTRβ polypeptide can be detected using whole-genome resequencing ortarget region resequencing (the latter also known as targetedresequencing) using suitably selected sources of DNA and polymerasechain reaction (PCR) primers in accordance with methods well known inthe art. See, for example, Bentley (2006) Curr Opin Genet Dev.16:545-52, and Li et al. (2009) Genome Res 19:1124-32. The methodtypically and generally entails the steps of genomic DNA purification,PCR amplification to amplify the region of interest, cycle sequencing,sequencing reaction cleanup, capillary electrophoresis, and dataanalysis. High quality PCR primers to cover region of interest aredesigned using in silico primer design tools. Cycle sequencing is asimple method in which successive rounds of denaturation, annealing, andextension in a thermal cycler result in linear amplification ofextension products. The products are typically terminated with afluorescent tag that identifies the terminal nucleotide base as G, A, T,or C. Unincorporated dye terminators and salts that may compete forcapillary eletrophoretic injection are removed by washing. Duringcapillary electrophoresis, the products of the cycle sequencing reactionmigrate through capillaries filled with polymer. The negatively chargedDNA fragments are separated by size as they move through the capillariestoward the positive electrode. After electrophoresis, data collectionsoftware creates a sample file of the raw data. Using downstreamsoftware applications, further data analysis is performed to translatethe collected color data images into the corresponding nucleotide bases.Alternatively or in addition, the method may include the use ofmicroarray-based targeted region genomic DNA capture and/or sequencing.Kits, reagents, and methods for selecting appropriate PCR primers andperforming resequencing are commercially available, for example, fromApplied Biosystems, Agilent, and NimbleGen (Roche Diagnostics GmbH). Foruse in the instant invention, PCR primers may be selected so as toamplify, for example, at least a relevant portion of a nucleic acidsequence encoding a mutant TRβ polypeptide having one or more mutationsat amino acid positions 234, 243, 316, and 317 of SEQ ID NO: 1.

Alternatively or in addition, a nucleic acid sequence encoding a mutantTRβ polypeptide may be detected using a Southern blot in accordance withmethods well known in the art.

In certain embodiments, the methods of the invention comprise the stepof performing an assay to detect a mutant of TRβ in a sample from asubject. As used herein, a “sample from a subject” refers to anysuitable sample containing cells or components of cells obtained orderived from a subject. In one embodiment the sample is a blood sample.In one embodiment the sample is a biopsy sample obtained from, forexample, the thyroid gland.

The disclosure also provides a ligand-mutant TRβ complex comprising: amutant TRβ polypeptide and a compound of Formula (IV). For example, themutant TRβ polypeptide forming the complex comprises one or moremutations at amino acid positions 234, 243, 316, and 317 of SEQ IDNO: 1. For example, the compound forming the complex is Compound A.

In addition, the disclosure provides a primer-nucleic acid complexcomprising: a mutant TRβ nucleic acid sequence, and a PCR primer that iscomplementary to the mutant TRβ nucleic acid sequence, wherein themutant nucleic acid sequence comprises an EZH2 gene mutation in anucleic acid sequence as defined in SEQ ID NO: 2.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties. However, where apatent, patent application, or publication containing expressdefinitions is incorporated by reference, those express definitionsshould be understood to apply to the incorporated patent, patentapplication, or publication in which they are found, and not to theremainder of the text of this application, in particular the claims ofthis application.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow, are intendedto illustrate and not limit the scope of the invention. It will beunderstood by those skilled in the art that various changes may be madeand equivalents may be substituted without departing from the scope ofthe invention, and further that other aspects, advantages andmodifications will be apparent to those skilled in the art to which theinvention pertains.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

EXAMPLES

Unless otherwise specified, the analytical instruments and parametersused for compounds described in the Examples are as follows:

The XRPD data were collected on X-Ray Powder Diffractometer (CubiX-ProXRD) with Cu Kα radiation (45 kV, 40 mA) from 3 to 45 degrees 2-theta(2θ) at a scanning rate of 0.12 degrees/min and step size of 0.020degrees.

Sample was placed on Si zero-return ultra-micro sample holders. Analysiswas performed using a 10 mm irradiated width and the followingparameters were set within the hardware/software:

-   X-ray tube: Cu KV, 45 kV, 40 mA-   Detector: X′Celerator-   ASS Primary Slit: Fixed 1°-   Divergence Slit (Prog): Automatic—5 mm irradiated length-   Soller Slits: 0.02 radian-   Scatter Slit (PASS): Automatic—5 mm observed length-   Scan Range: 3.0-45.0°-   Scan Mode: Continuous-   Step Size: 0.02°-   Time per Step: 10 s-   Active Length: 2.54°    Following analysis the data was converted from adjustable to fixed    slits using the X′Pert HighScore Plus software with the following    parameters:-   Fixed Divergence Slit Size: 1.00°, 1.59 mm-   Crossover Point: 44.3° Omega

In the Examples described below, unless otherwise specified, Compound 4is the benzoyl protected compound.

Example 1 Preparation ofN-(3,5-dichloro-4-((6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)benzamide(Compound 4 where R² is benzoyl)

A 1 L, three-neck, round-bottom flask equipped with overhead stirring, athermocouple, reflux condenser, and N₂ inlet/outlet was charged with3,6-dichloropyridazine (100 g, 0.672 mol, 1 wt),4-amino-2,6-dichlorophenol (122 g, 0.686 mol, 1.02 equiv), and DMAC (500mL, 5 vol). The resulting solution was charged with cesium carbonate(251 g, 0.771 mol, 1.15 equiv) and the suspension was heated to 110° C.After 3 h at that temperature, the batch temperature was lowered to 70°C. and stirred at that temperature for 16 h. ¹H NMR analysis (DMSO)showed nearly all the dichloropyridazine had been consumed and thereaction was deemed complete. The batch was cooled to room temperatureand transferred to a 3 L, round-bottom flask with the aid of EtOAc (2 L,20 vol). Silica gel (100 g, 1 wt) was added and the suspension wasagitated for 30 min and filtered. The reactor and cake were rinsed withEtOAc (500 mL, 5 vol) until the filtrate eluted colorless. The resultingfiltrate was treated with 10% aqueous NaCl (2 L, 20 vol), the biphasicmixture was agitated for 30 min, and the lower aqueous layer wasdiscarded. The upper organic layer was concentrated to dryness underreduced pressure. EtOAc (100 mL, 1 vol) was added to the residue andconcentrated to dryness under reduced pressure to provide crude Compound2 (251 g, 128% yield) as an oil. HPLC analysis showed a purity of 93.4%.¹H NMR analysis (DMSO) was consistent with the assigned structure andshowed ≈25% DMAC and 2% EtOAc present.

Other conditions for synthesizing Compound 2 are described in Tables 1-3below.

TABLE 1 Summary of Reaction Parameters for Compound 2 Scale ¹H NMR or(g) Conditions % Yield HPLC 5.0 Int. 1 1 equiv 80 ≈90% pure DMSO 5 volKOtBu 1.1 equiv Int. 3 1 equiv 85° C. 15 Int. 1 1 equiv 84 ≈90% pureDMSO 5 vol KOtBu 1.1 equiv Int. 3 1 equiv 85° C. 15 Int. 1 1 equiv 70≈90% pure DMAC 5 vol KOtBu 1.1 equiv Int. 3 1 equiv 85° C. 50.0 Int. 10.98 equiv — Telescoped DMAC 5 vol KOtBu 1.1 equiv Int. 3 1 equiv 85° C.17.45 Int. 1 0.98 equiv 85 ≈95% DMSO 5 vol KOtBu 1.1 equiv Int. 3 1equiv 85° C.

TABLE 2 Summary of Reaction Parameters for Compound 2 HPLC Time % AUCNMR Solvent Conditions (h) Yield (220 nm) Purity DMSO 1.15 + 0.26 equiv26 98 72.2 Contains KOtBu DMSO 1 equiv Int. 1 1.02 equiv Int. 3 85° C.DMAC 1.15 equiv Cs₂CO₃ 2 130 88.8 Contains 1 equiv Int. 1 33% 1.02 equivInt. 3 DMAC 120° C. NMP 1.15 equiv Cs₂CO₃ 2 172 86.8 Contains 1 equivInt. 1 46% 1.02 equiv Int. 3 NMP 120° C. DMAC 1.0 equiv Cs₂CO₃ 4 12066.0 Contains 1 equiv Int. 1 42% 1.02 equiv Int. 3 DMAC 120° C. DMAC1.02 equiv 2 1 128    93.4% Contains 1.15 equiv Cs₂CO₃ 2.25 26% 5 volDMAC 3.25 DMAC 110° C. to 70° C. 19  2% EtOAc

TABLE 3 Summary of Reaction Parameters for Compound 2 (all reactions arein DMAC) Temp. HPLC IPC Base Time (h) ° C. (220 nm) Cs₂CO₃ 3 110 94.8%15 90 94.4% Li₂CO₃ 3 110 12.7% K₂CO₃ 3 110 91.6% 15 90 91.4% Na₂CO₃ 3110 84.5% 15 90 84.9% NaOAc 3 110 25.2% KF 3 110 54.1% DIPEA 3 110 22.8%DBU 3 110 80.8% 15 90 — DABCO 3 110  6.2% KOH 3 110 85.1% (ground)

The crude 2 above was taken up in acetic acid (1.48 L, 7.5 vol) andbenzoic anhydride (168 g, 0.741 mol, 1.1 equiv) was added. The resultingmixture was heated to 100° C. and after 35 min at that temperature, theamount of 2 was 0.8%. Sodium acetate (110 g, 2 equiv) was added and thetemperature increased to 110° C. After 14.5 h at that temperature, HPLCanalysis of the reaction mixture showed no intermediate remaining, andthe reaction was deemed complete. The batch was cooled to 75° C. andwater (1.5 L, 7.7 vol) was added over a period of 1 hour whilemaintaining a batch temperature between 72-75° C. The batch was cooledto 21° C. and filtered through Sharkskin filter paper. The reactor andcake were washed sequentially with water (1 L, 5 vol). After drying thecollected solid in a 50° C. vacuum oven for 16 h, the yield of crude 4was 195 g (77%). HPLC analysis (Method B, 220 nm) showed a purity of91.6%.

HPLC method B:   Column: Waters Sunfire C18, 3.5 μM, 4.6 × 150 mm Flowrate: 1.0 mL/min. Mobile phase A: 0.05% TFA in water Mobile phase B:0.05% TFA in H2O Diluent: 50:50 MeCN/H₂O Time (min.) % A % B 0.0 98 25.0 98 2 20 5 95 25 5 95 25.1 98 2 30 98 2

¹H NMR analysis (DMSO) was consistent with the assigned structure andindicated an acetic acid content of 1%. Benzoyl chloride was also usedfor the protection instead of benzoic anhydride. When benzoyl chloridewas used, bases such as cesium carbonate or potassium carbonate wereused and the reaction was carried out at room temperature.

Other conditions for synthesizing Compound 4 are described in Tables 4and 5 below.

TABLE 4 Protection/hydrolysis of Compound 2 (Yields Reported fromInt. 1) (benzoyl protecting group) HPLC Time IPC % Purity SolventConditions (h) % Pdt Yield (% AUC) Acetic acid 1. 1.03 equiv2 1. 2h   1. 70   71 78.2  1.15 equiv Cs₂CO₃ 2. 19 h   2. 70.7  3 vol DMSO 3.20.5 h 3. 68.8 2. Bz₂O 3. Acetic acid, 115° C. Acetic acid 1.1 equivBz₂O 16.5 79.0 66 91.6 2 equiv NaOAc 110° C. Acetic acid 1.1 equiv Bz₂O14.25 76.6 77 91.6 2 equiv NaOAc 100-110° C.

TABLE 5 Summary of Reaction Parameters for Compound 4(acetate protectinggroup) Scale ¹H NMR or (g) Conditions % Yield HPLC  5.0 NaOAc 2 equiv 76≈95% Acetic acid 4 vol 115° C. 15.0 NaOAc 2 equiv 60 >99% Acetic acid 4vol 115° C. 50.0 g NaOAc 2 equiv 51 ≈95% (Int. 1) Acetic acid 4 vol2-step 115° C.

Purification of Compound 4: A 5 L, three-neck, round-bottom flaskequipped with overhead stirring, a thermocouple, reflux condenser, andN₂ inlet/outlet was charged with crude 4 (100 g, 1 wt) and acetic acid(2 L, 20 vol). The slurry was agitated and heated to 95° C., anddissolution occurred. Water (2 L, 20 vol) was added over a period of2.75 h while maintaining a batch temperature of ≈95° C., andprecipitation occurred. The resulting slurry was heated at 95° C. foranother 30 min before heating was removed. After the batch reachedambient temperature, it was stirred at that temperature overnight forconvenience and filtered through Sharkskin filter paper. The reactor andcake were rinsed sequentially with water (1 L, 10 vol). The collectedwhite solid was dried in a 40° C. vacuum oven to a constant weight of 91g (91%). HPLC analysis of the dried solid showed a purity of 98.0%. ¹HNMR analysis (DMSO) was consistent with the assigned structure andshowed an acetic acid content of 0.3%. Table 6 below lists otherconditions for purifying Compound 4.

TABLE 6 Purification of Compound 4 (R² = Bz) HPLC Time % Purity SolventConditions (h) Yield (% AUC) Acetic 1 equiv 4 1 90 96.8 acid/ 20 volAcOH H₂O 20 vol water 88-100° C. Acetic 1 equiv 4 acid/ 20 vol AcOH 3 9198.0 H₂O 20 vol water 95° C. Acetic 1 equiv 4 4 92 98.0 acid/ 20 volAcOH H₂O 20 vol water 95° C. Acetic 1 equiv 4 1 90 98.9 acid/ 12 volAcOH H₂O 10 vol water 100-110° C.

Example 2 Preparation of6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one (Int. 7)

A 4 L, four-neck, round-bottom flask equipped with overhead stirring, athermocouple, N₂ inlet/outlet, and a reflux condenser was charged with 4(95 g, 0.253 mol, 1 wt), THF (665 mL, 7 vol), and LiCl (32.3 g, 0.759mol, 3 equiv). The resulting suspension was heated to 35° C., andisopropenylmagnesium bromide solution (0.5 M in THF, 1.72 L, 0.859 mol,3.4 equiv) was added over a period of 80 min while maintaining a batchtemperature between 35-45° C. After heating the resulting slurry at 40°C. for 3 h, HPLC analysis showed a conversion of 87%). Additionalisopropenylmagnesium bromide solution (0.5 M in THF, 51 mL, 0.026 mol,0.1 equiv) was added and the slurry was agitated at 40-43° C. foranother 90 min. HPLC analysis showed a conversion of 92.9% and thereaction was deemed complete. The heating was removed, the reactionmixture was cooled to 14° C., and 3 N aqueous HCl (380 mL, 4 vol) wasadded slowly over 15 min while maintaining a batch temperature below 26°C., after which time all solids had dissolved. The lower aqueous layerwas removed and extracted with THF (350 mL, 3.7 vol). After removing thelower aqueous layer, the combined organic layers were concentrated underreduced pressure to approximately 5 vol with respect to 4. The resultingsolution was charged with 10% (w/w) aqueous KOH (532 mL, 5.6 vol), andthe mixture was heated to 85° C. while distilling off THF using ashort-path distillation apparatus. The batch was held at 85° C. for 11h, and the heating was removed. The batch was cooled to ambienttemperature overnight for convenience. HPLC analysis (Method A below) ofthe resulting slurry showed a conversion of 99% to Int. 7 and thereaction was deemed complete.

HPLC method A   Column: Waters Sunfire C18, 3.5 μM, 4.6 × 150 mm Flowrate: 1.0 mL/min. Mobile phase A: 0.05% TFA in water Mobile phase B:0.05% TFA in H₂O Diluent: 50:50 MeCN/H₂O Time (min.) % A % B 0.0 98 215.0 5 95 25 5 95 25.1 98 2 30 98 2

The batch temperature was adjusted to 48° C. and 3 N aqueous HCl (152mL, 1.6 vol) was added over 35 min to adjust the pH to 7.5-8.0 whilemaintaining a batch temperature of 46-48° C. Heating was removed and theslurry was cooled to 30° C. ¹H NMR analysis (DMSO) showed an Int. 7/THFmol ratio of 1.0:0.22 (Attachment 14). The batch was filtered at 30° C.through Sharkskin filter paper and the reactor and cake were washed withwater (475 mL, 5 vol) sequentially. The beige solid Int. 7 was dried ina 40° C. vacuum oven to a constant weight of 81.6 g (102% yield). KarlFischer analysis indicated a water content of 0.8%. ¹H NMR (DMSO) wasconsistent with the assigned structure and indicated a THF content of0.4%. HPLC analysis showed a purity of 92.6%. Tables 7-10 below providesummaries of reaction parameters for producing Int. 7.

TABLE 7 Summary of Grignard Isopropenylation Runs Scale (g) Conditions %Yield ¹H NMR or HPLC 5.0 1 equiv 4 97 94.1 7 vol THF 3.4 equiv Grignard3 equiv LiCl 40° C. 25.0 1 equiv 4 102 93.1 7 vol THF 3.4 equiv Grignard3 equiv LiCl 40° C. 95.5 1 equiv 4 101 92.6 7 vol THF 3.5 equiv Grignard3 equiv LiCl 40° C. 5.0 1 eq 4 97 90.7 3 eq LiCl 8 vol THF 3.4 eqGrignard 1.5M in MeTHF 40° C. 5.0 1 eq 4 100 87.5 3 eq LiCl 15 vol THF 2eq t-BuMgCl 2M in THF 1.7 eq Grignard 1.5M in MeTHF 40° C. 5.0 1 eq 4 9086.9 3 eq LiCl 15 vol THF 3.6 eq Grignard 0.5M in THF 40° C. 10.0 1 eq 4114 85.4 3 eq LiCl 13 vol THF 3.7 eq Grignard 1.5M in MeTHF 40° C. 10.01 eq 4 67 89.3 3 eq LiBr 13 vol THF 3.7 eq Grignard 1.5M in MeTHF 40° C.10.0 1 eq 4 88 91.2 5 eq LiCl 13 vol THF 3.7 eq Grignard 0.5M in THF 40°C.

TABLE 8 Summary of Grignard Isopropylation Runs Scale % ¹H NMR or (g)Conditions Yield HPLC 1.0 1 equiv 4 (R² = Ac) 35 >95 20 vol THF 3.3equiv iPrMgCl 30° C. 5.0 1 equiv 4 (R² = Ac) 94 ≈90 20 vol THF 6 equiviPrMgCl 40° C. 2.0 1 equiv 4 51 >95 20 vol THF 4 equiv iPrMgCl 20° C.1.21 1 equiv 4 — — 20 vol Dioxane 4.1 equiv iPrMgCl 40° C. 2.0 1 equiv 4(R² = Ac) — — 8 equiv iPrMgCl 30 vol THF 25-42° C. 1.1 1 equiv 4 —Telescoped 2 equiv LiCl into 4 equiv iPrMgCl oxidation 27 vol THF 1.0 1equiv 4 (R² = Ac) — — 3 equiv LiCl 5 equiv iPrMgCl 25 vol THF 2.0 1equiv 4 46 >95 3 equiv LiCl 4 equiv iPrMgCl 10 vol THF 4.0 1 equiv 4 95  88 3 equiv LiCl 4.1 equiv iPrMgCl 7 vol THF 5.0 1 equiv 4 — Telescoped3 equiv LiCl into 3.5 equiv iPrMgCl oxidation 7 vol THF 10.0 1 equiv 4 —Telescoped 3 equiv LiCl into 3.2 equiv iPrMgCl oxidation 7 vol THF 5.0 1equiv 4 — Telescoped 3 equiv LiCl into 3.4 equiv iPrMgCl oxidation 10vol THF 5.0 1 equiv 4 — Telescoped 3 equiv LiCl into 3.4 equiv iPrMgCloxidation 10 vol THF

TABLE 9 Summary of the Bromine Oxidation of Pyridazinone Compound 5Scale (g) Conditions % Yield ¹H NMR or HPLC 0.13 5 (R² = Ac) 1 equiv 82≈95% Br₂ 2 equiv AcOH 10 vol 90° C. 3.09 5 (R² = Ac) 1 equiv 84 ≈80% Br₂1.5 equiv AcOH 7 vol 90° C. 0.82 5 (R² = Bz) 1 equiv 84 >95% Br₂ 1.5equiv AcOH 7 vol 90° C. 1.1 5 (R² = Bz) 1 equiv 86 ≈90    AcOH 10 vol2-step 2-step Br₂ 5 equiv 90° C. 1.02 5 (R² = Bz) 1 equiv 100 ≈95   AcOH 10 vol Br₂ 1.5 equiv 90° C. 1.55 5 (R² = Bz) 1 equiv 103  89.6 AcOH10 vol 2-step 2-step Br₂ 1.5 equiv 60° C. 1.71 5 (R² = Bz) 1 equiv 84 91.9 AcOH 10 vol 2-step Br₂ 1.5 equiv 60° C.

TABLE 10 Summary of Deprotection of Compound 6 to obtain Int. 7Protecting Time Conversion group Conditions Temp (h) (% AUC) Acetyl TFA(10 vol) 90° C. 15 21.6 Water (10 vol) 61 19.9 Bz TFA (10 vol) 90° C. 1540.1 (benzoyl) Water (10 vol) 61 100 Bz BF₃•Et₂O (6 equiv) RT 15 NR MeOH(10 vol) 60° C. 4.5 13.0 Add water (10 equiv) 60° C. 18 31.1 Bz 6N KOH(10 vol) 90° C. 16 100 Bz 2N NaOH (5 vol) RT 5.5 4.7 60° C. 16 31.2 2NNaOH (2.5 vol) RT 5.5 6.1 Bz MeOH (2.5 vol) 60° C. 16 40.3 Bz Na₂CO₃ (10wt %, 70° C. 2 7.0 5 vol) Bz KOH (10 wt %, 70° C. 2 19.6 5 vol) 23 86.952 97.3

Example 3 Preparation of(Z)-ethyl(2-cyano-2-(2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)hydrazono)acetyl)carbamate(Int. 8)

A 2 L, three-neck, round-bottom flask equipped with overhead stirring, athermocouple, N₂ inlet/outlet was charged with Int. 7 (75.0 g, 0.239mol, 1 wt), acetic acid (600 mL, 8 vol), water (150 mL, 2 vol), andconcentrated HCl (71.3 mL, 0.95 vol). The resulting thin slurry wascooled to 6° C. and a solution of NaNO₂ (16.8 g, 0.243 mol, 1.02 equiv)in water (37.5 mL, 0.5 vol) was added over a period of 10 min whilemaintaining a batch temperature below 10° C. After an additional 10 minof agitation between 5-10° C., HPLC analysis showed complete conversionof Int. 7 to the diazonium intermediate. A solution of NaOAc (54.5 g,0.664 mol, 2.78 equiv) in water (225 mL, 3 vol) was added over a periodof 6 min while maintaining a batch temperature below 10° C.N-cyanoacetylurethane (37.9 g, 0.243 mol, 1.02 equiv) was immediatelyadded, the cooling was removed, and the batch naturally warmed to 8° C.over 35 min. HPLC analysis showed complete consumption of the diazoniumintermediate and the reaction was deemed complete. The batch warmednaturally to 21° C. and was filtered through Sharkskin filter paper. Thereactor and cake were washed sequentially with water (375 mL, 5 vol)twice. The collected orange solid was dried in a 35° C. vacuum oven for64 h to provide crude Int. 8 (104.8 g, 91%).

A 1 L, three-neck, round-bottom flask equipped with overhead stirring, athermocouple, and N₂ inlet/outlet was charged with crude Int. 8 (104.4g, 1 wt) and acetic acid (522 mL, 5 vol). The resulting slurry washeated to 50° C. and held at that temperature for 1.5 h. The batchcooled naturally to 25° C. over 2 h and was filtered through Sharkskinfilter paper. The reactor and cake were washed sequentially with water(522 mL, 5 vol) and the cake conditioned under vacuum for 1.75 h. Thelight orange solid was dried to constant weight in a 40° C. vacuum ovento provide 89.9 g (78% from Int. 7) of the desired product. ¹H NMR(DMSO) was consistent with the assigned structure.

Example 4 Preparation of2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(Compound A)

A 2 L, three-neck, round-bottom flask equipped with overhead stirring, athermocouple, N₂ inlet/outlet, and reflux condenser was charged withInt. 8 (89.3 g, 0.185 mol, 1 wt), DMAC (446 mL, 5 vol), and KOAc (20.0g, 0.204 mol, 1.1 equiv). The mixture was heated to 120° C. and held atthat temperature for 2 h. HPLC analysis showed complete conversion toCompound A. The batch temperature was adjusted to 18° C. over 1 h, andacetic acid (22.3 mL, 0.25 vol) was added. The batch temperature wasadjusted to 8° C., and water (714 mL, 8 vol) was added over 1 h; anorange slurry formed. The batch was filtered through Sharkskin filterpaper and the cake was allowed to condition overnight under N₂ withoutvacuum for convenience. A premixed solution of 1:1 acetone/water (445mL, 5 vol) was charged to the flask and added to the cake as a rinsewith vacuum applied. After 2 h of conditioning the cake under vacuum, itwas transferred to a clean 1 L, three-neck, round-bottom flask equippedwith overhead stirring, a thermocouple, and N₂ inlet/outlet. Ethanol(357 mL, 4 vol) and acetone (357 mL, 4 vol) were charged and theresulting slurry was heated to 60° C.; dissolution occurred. Water (890mL, 10 vol) was added over a period of 90 min while maintaining a batchtemperature between 55-60° C. The resulting slurry was allowed to coolto 25° C. and filtered through Sharkskin filter paper. The reactor andcake were washed sequentially with a solution of 1:1 EtOH/water (446 mL,5 vol). The cake was conditioned overnight under N₂ without vacuum forconvenience. The cracks in the cake were smoothed and vacuum applied.The cake was washed with water (179 mL, 2 vol) and dried in a 45° C.vacuum oven to a constant weight of 70.5 g (87%, crude Compound A). HPLCanalysis showed a purity of 94.8%.

A 500 mL, three-neck, round-bottom flask equipped with overheadstirring, a thermocouple, N₂ inlet/outlet, and reflux condenser wascharged with crude Compound A (70.0 g) and MIBK (350 mL, 5 vol). Theorange slurry was heated to 50° C. and held at that temperature for 2 h.The batch cooled naturally to 23° C. and was filtered through Sharkskinfilter paper. The reactor and cake were washed sequentially with MIBK(35 mL, 0.5 vol) twice. The collected solids were dried in a 45° C.vacuum oven to a constant weight of 58.5 g (84%). This solid was chargedto a 500 mL, three-neck, round-bottom flask equipped with overheadstirring, a thermocouple, N₂ inlet/outlet, and reflux condenser. Ethanol(290 mL, 5 vol) was added and the slurry was heated to reflux. After 3.5h at reflux, XRPD showed the solid was consistent with Form I, andheating was removed. Upon reaching 25° C., the batch was filteredthrough filter paper, and the reactor and cake were washed sequentiallywith EtOH (174 mL, 3 vol). The tan solid Compound A was dried in a 40°C. vacuum oven to a constant weight of 50.4 g (87%, 64% from Int. 8).HPLC analysis showed a purity of 99.1%. ¹H NMR (DMSO) was consistentwith the assigned structure.

Example 5 Scaled up preparation of2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(Compound A)

A larger scale batch of Compound A was synthesized according to thescheme below. The conditions in the scheme below are similar to thosedescribed in Examples 1-4 above.

Synthesis of 4

A 50 L jacketed glass vessel (purged with N₂) was charged with3,6-dichloropyridazine (2.00 kg), 4-amino-2,6-dichlorophenol (2.44 kg)and N,N-dimethylacetamide (10.0 L). The batch was vacuum (26inHg)/nitrogen (1 PSIG) purged 3 times. Cesium carbonate (5.03 kg) wasadded and the batch temperature was adjusted from 22.3° C. to 65.0° C.over 3.5 hours. The batch was held at 65.0° C. for 20 hours. At thispoint, ¹H NMR analysis indicated 3.34% 3.6-dichloropyridazine relativeto 2. The batch temperature was adjusted to 21.5° C. and ethyl acetate(4.00 L) was added to the batch. The batch was agitated for 10 minutesand then filtered through a 18″ Nutsche filter equipped withpolypropylene filter cloth. The filtration took 15 minutes. Ethylacetate (5.34 L) was charged to the vessel and transferred to the filteras a rinse. The batch was then manually re-suspended in the filterbefore re-applying vacuum. This process was repeated 2 more times andthe filter cake was conditioned for 10 minutes. The filtrate was chargedto a 100-L vessel that contained (16.0 L) of a previously prepared 15%sodium chloride in H₂O. The batch was agitated for 5 minutes and thenallowed to separate for 35 minutes. The interface was not visible, sothe calculated 23 L of the lower aqueous phase was removed. 16.0 L of15% Sodium chloride in H₂O was added to the batch. The batch wasagitated for 6 minutes and then allowed to separate for 7 minutes. Theinterface was visible at ˜19 L and the lower aqueous phase was removed.17.0 L of 15% Sodium chloride in H₂O was added to the batch. The batchwas agitated for 7 minutes and then allowed to separate for 11 minutes.The lower aqueous phase was removed. The vessel was set up for vacuumdistillation and the batch was concentrated from 17.0 L to 8.0 L over 2hours 20 minutes with the batch temperature kept around 21° C. Benzoicanhydride (3.19 kg) and acetic acid (18.0 L) were charged to the vessel.The vessel was set up for vacuum distillation and the batch wasconcentrated from 28.0 L to 12.0 L over 2 days (overnight hold at 20°C.) with the batch temperature kept between 20 and 55° C. At this point,¹H NMR analysis indicated a mol ratio of acetic acid to ethyl acetate of1.0:0.015. Acetic acid (4.0 L) was charged to the batch and the batchwas distilled to 12 L. ¹H NMR analysis indicated a mol ratio of aceticacid to ethyl acetate of 1.0:0.0036. Acetic acid (20.0 L) was charged tothe batch and the batch temperature was adjusted to 70.0° C. The batchwas sampled for HPLC analysis and 2 was 0.16%. Sodium acetate (2.20 kg)was added to the batch and the batch temperature was adjusted from 72.4°C. to 110.0° C. After 18.5 hours, HPLC analysis indicated no Int. Bdetected. The batch temperature was adjusted from 111.3 to 74.7° C. andDI water (30.0 L) was added to the batch over 2 hours. The batchtemperature was adjusted to 20.5° C. and then filtered using a 24″Haselloy Nutsche filter equipped with polypropylene filter cloth. Apreviously prepared solution of 1:1 acetic acid in DI H₂O (10.0 L) wascharged to the vessel and agitated for 5 minutes. The wash wastransferred to the filter and the batch was then manually re-suspendedin the filter before re-applying vacuum. DI H₂O (10.0 L) was charged tothe vessel and then transferred to the filter. The batch was manuallyre-suspended in the filter before re-applying vacuum. DI H₂O (10.0 L)was charged directly to the filter and the batch was then manuallyre-suspended in the filter before re-applying vacuum. The filter cakewas allowed to condition for 18 hours to give 14.4 kg of 4. HPLCanalysis indicated a purity of 93.7%. This wet cake was carried forwardinto the purification. A 100 L jacketed glass vessel (purged with N₂)was charged with crude 4 (wet cake 14.42 kg), acetic acid (48.8 L) andthe agitator was started. DI H₂O (1.74 L) was charged. The batch (aslurry) temperature was adjusted from 18.1 to 100.1° C. over 4.25 hours.The batch was held at 100.1 to 106.1° C. for 1 hour and then adjusted to73.1° C. DI H₂O (28.0 L) was added to the batch over 1 hour keeping thebatch temperature between 73.1 and 70.3° C. The batch temperature wasadjusted further from 70.3° C. to 25.0° C. overnight. The batch wasfiltered using a 24″ Hastelloy Nutsche filter equipped withpolypropylene filter cloth. The filtration took 13 minutes. A solutionof DI H₂O (9.00 L) and acetic acid (11.0 L) was prepared and added tothe 100 L vessel. The mixture was agitated for 5 minutes and thentransferred to the filter cake. DI H₂O (20.0 L) was charged to thevessel, agitated for 6 minutes and then transferred to the filter cake.DI H₂O (20.0 L) was charged to the vessel, agitated for 9 minutes andthen transferred to the filter cake. The batch was allowed to conditionfor 3 days and then transferred to drying trays for vacuum oven drying.After 3 days at 50° C. and 28″/Hg, the batch gave a 74% yield (3.7 kg)of 4 as an off-white solid. The ¹H NMR spectrum was consistent with theassigned structure, HPLC analysis indicated a purity of 98.87% and KFanalysis indicated 0.14% H₂O.

Synthesis of Int. 7

A 100-L jacketed glass vessel (purged with N2) was charged withtetrahydrofuran (44.4 L). The agitator was started (125 RPM) and 4 (3.67kg) was charged followed by lithium chloride (1.26 kg). The batchtemperature was observed to be 26.7° C. and was an amber solution.Isopropenylmagnesium bromide 1.64 molar solution in 2-methyl THF (21.29kg) was added over 2½ hours keeping the batch between 24.3 and 33.6° C.The batch was agitated at 24.5° C. for 17 hours at which point HPLCanalysis indicated 9% 4. A 2nd 100-L jacketed glass vessel (purged withN2) was charged with 3N hydrogen chloride (18.3 L). The batch wastransferred to the vessel containing the 3N HCl over 25 minutes keepingthe batch temperature between 20 and 46° C. A bi-phasic solution wasobserved. The quenched batch was transferred back to the 1^(st) 100-Lvessel to quench the small amount of residue left behind. THF (2.00 L)was used as a rinse. The batch temperature was observed to be 40.9° C.and was agitated at 318 RPM for 45 minutes. The batch temperature wasadjusted to 21.8° C. and the layers were allowed to separate. Theseparation took 10 minutes. The lower aqueous phase was removed (˜26.0L). A solution of sodium chloride (1.56 kg) in DI water (14.0 L) wasprepared and added to the batch. This was agitated at 318 RPM for 10minutes and agitator was stopped. The separation took 3 minutes. Thelower aqueous phase was removed (˜16.0 L). The batch was vacuumdistilled from 58.0 L to 18.4 L using ˜24″/Hg and a jacket temperatureof 50 to 55° C. A solution of potassium hydroxide (2.30 kg) in DI water(20.7 L) was prepared in a 72-L round bottom flask. The vessel was setup for atmospheric distillation using 2 distillation heads and the batchwas transferred to the 72-L vessel. THF (0.75 L) was used as a rinse.The batch volume was ˜41.0 L, the temperature was adjusted to 64.1° C.and distillation started with the aid of a N2 sweep. Heating wascontinued to drive the batch temperature to 85.4° C. while distilling atwhich point the 72-L vessel was set up for reflux (batch volume wasabout 28.0 L at the end of the distillation). The batch was held at 85°C. for 13 hours at which point HPLC analysis indicated 0.3% compound 6A.Heating was stopped and the batch was transferred to a 100-L jacketedglass vessel. Solids were observed. The batch temperature was adjustedfrom 70.6° C. to 56.7° C. A previously prepared solution of sodiumhydrogen carbonate (2.82 kg) in DI water (35.0 L) was added over 80minutes keeping the batch temperature between 56.7 and 46.7° C. Thebatch pH at the end of the addition was 9.8. The batch was held at 46.7to 49.0° C. for 40 minutes and then cooled to 25.0° C. The batch wasfiltered using a 18″ stainless steel Nutsche filter. DI water (18.4 L)was charged to the vessel and transferred to the filter. The filter cakewas manually re-suspended in the filter and then the liquors wereremoved. This process was repeated once more and the filter cake was 3″thick. The filter cake was conditioned on the filter for 3 days, wastransferred to drying trays and dried in a vacuum oven at 45° C. toprovide 2.93 kg Int. 7 (95% yield) with an HPLC purity of 87.6%.

Synthesis of Int. 8

A 100 L jacketed glass vessel (purged with N₂ and plumbed to a causticscrubber) was charged with acidic acid (13.0 L). Int. 7 (2.85 kg) wascharged to the vessel and the agitator was started.N-Cyanoacetylurethane (1.56 kg) and DI water (5.70 L) were charged tothe vessel. The batch temperature was adjusted from 17.0° C. to 5.5° C.and a thin slurry was observed. At this point 37% hydrogen chloride(2.70 L) was added over 10 minutes keeping the batch temperature between4.8° C. and 8.8° C. A previously prepared solution of sodium nitrite(638 g) in DI water (1.42 L) was added over 26 minutes keeping the batchtemperature between 5.8° C. and 8.7° C. A brown gas was observed in thevessel head space during the addition. HPLC analysis indicated no Int. 7detected. At this point a previously prepared solution of sodium acetate(2.07 kg) in DI water (8.50 L) was added over 47 minutes keeping thebatch temperature between 5.5° C. and 9.5° C. After the addition, a thinlayer of orange residue was observed on the vessel wall just above thelevel of the batch. The batch temperature was adjusted from 9.4° C. to24.5° C. and held at 25° C. (±5° C.) for 12 hours. The batch wasfiltered using a 24″ Hastelloy Nutsche filter equipped with tight-weavepolypropylene filter cloth. The filtration took 30 minutes. The vesselwas rinsed with 14.3 L of a 1:1 acidic acid/DI water. The orange residueon the reactor washed away with the rinse. The rinse was transferred tothe filter where the batch was manually re-suspended. Vacuum wasre-applied to remove the wash. A 2^(nd) 1:1 acidic acid/DI water washwas performed as above and the batch was conditioned on the filter for26 hours. HPLC analysis of the wet filter cake indicated purity was90.4%. The batch was dried to a constant weight of 3.97 kg (91% yield)in a vacuum oven at 45° C. and 28″/Hg.

Preparation of Compound a DMAC Solvate

A 100 L, jacketed, glass vessel purged with N₂ was charged with Int. 8(3.90 kg) and potassium acetate (875 g). N,N-dimethylacetamide (DMAC,18.3 L) was charged to the vessel and the agitator was started. Thebatch temperature was adjusted to 115° C. over 2 h. After 2 h at 115°C., the batch was sampled and HPLC analysis indicated 0.27% Int. 8remained. The batch temperature was adjusted to 25.0° C. overnight.Acetic acid (975 mL) was added to the batch and the batch was agitatedfurther for 3 h. The batch was transferred to a carboy and the vesselwas rinsed clean with 800 mL of DMAC. The batch was transferred back tothe 100 L vessel using vacuum through a 10 μm in-line filter and a DMACrinse (1.15 L) was used. The filtration was fast at the beginning butslow at the end, plugging up the filter. The batch temperature wasadjusted to 11.1° C. and DI water (35.1 L) was added over 2 h 20 min,keeping the batch temperature between 5-15° C. The batch was held for 1h and filtered, using an 18″ Nutsche filter equipped with tight-weavepolypropylene cloth. The filtration took 15 h. A 1:1 ethanol/DI waterwash (19.5 L) was charged to the vessel, cooled to 10° C., andtransferred to the filter cake. The cake was allowed to condition underN₂ and vacuum for 8 h and transferred to drying trays. The batch wasdried in a vacuum oven at 45° C. and 28″/Hg to give 89% yield (3.77 kg)of Compound A DMAC solvate as an orange/tan solid. The ¹H NMR spectrumwas consistent with the assigned structure and Karl Fischer analysisindicated 0.49% H₂O. XRPD indicated the expected form, i.e., Compound ADMAC solvate. Thermogravimetric analysis (TGA) indicated 16% weightloss. HPLC analysis indicated a purity of 93.67%.

Preparation of Crude Compound A

A 100 L, jacketed, glass vessel purged with N₂ was charged with CompoundA DMAC solvate (3.75 kg) and ethanol (15.0 L). The agitator was startedand acetone (15.0 L) was added. The batch temperature was adjusted from10.6° C. to 60.0° C. over 1 h. At this point, the batch was in solution.DI water was added to the batch over 1.5 h, keeping the batchtemperature at 60±5° C. The batch was held at 60±5° C. for 1 h andcooled to 23.5° C. An 18″ Nutsche filter equipped with tight-weave (0.67CFM) polypropylene cloth was set up and the batch was filtered. Thefiltration took 15 h. A 1:1 ethanol/DI water wash (19.5 L) was chargedto the vessel and transferred to the filter cake. The cake was allowedto condition under N₂ and vacuum for 8 h and transferred to dryingtrays. The batch was dried in a vacuum oven at 45° C. and 28″/Hg forfive days to give a 94% yield (2.90 kg) of Compound A as a powdery tansolid. The ¹H NMR spectrum is consistent with the assigned structure andKarl Fischer analysis indicated 6.6% H₂O. XRPD indicated the expectedform of dihydrate. TGA indicated 6.7% weight loss. HPLC analysisindicated a purity of 96.4% (AUC).

Purification of Crude Compound A

A 50 L, jacketed, glass vessel purged with N₂ was charged with CompoundA crude (2.90 kg) and methyl isobutyl ketone (14.5 L). The agitator wasstarted and the batch temperature was adjusted from 20.2° C. to 50.4° C.over 1.5 h. The batch was held at 50° C. (±5° C.) for 1 h and cooled to20-25° C. The batch was held at 20-25° C. for 2.5 h. An 18″ Nutschefilter equipped with tight-weave (0.67 CFM) polypropylene cloth was setup and the batch was filtered. The filtration took 20 min. Methylisobutyl ketone (MIBK, 1.45 L) was charged to the vessel and transferredto the filter cake. The cake was manually resuspended and the liquorswere pulled through with vacuum. Methyl isobutyl ketone (2.90 L) wascharged to the filter cake and the cake was manually resuspended. Theliquors were pulled through with vacuum and the cake was conditionedwith vacuum and nitrogen for 15 h. The filter cake dried into a tan,hard 18″×1½″ disc. This was manually broken up and run through coffeegrinders to give a 76% yield (2.72 kg) of MGL-3196 MIBK solvate as atan, powdery solid. No oven drying was necessary. The ¹H NMR spectrumwas consistent with the assigned structure and Karl Fischer analysisindicated <0.1% H₂O. XRPD indicated the expected form MIBK solvate. TGAindicated 17.3% weight loss. HPLC analysis indicated a purity of 98.5%.

Example 6 Conversion of Compound A to Form I

Purified Compound A (4802 g) as a 1:1 MIBK solvate which was obtainedfrom Int. 8 as described in Example 5 above was added into a jacketed,100 L reactor along with 24 liters of ethanol. The resulting slurry washeated to 80±5° C. (reflux) over 1 h 25 min; the mixture was stirred atthat temperature for 4 h 25 min. Analysis of the filtered solids at 2 h55 min indicated that the form conversion was complete, with the XRPDspectra conforming to Form I. The mixture was cooled to 20±5° C. over 45min and stirred at that temperature for 15 min. The slurry was filteredand the filter cake was washed twice with prefiltered ethanol (2×4.8 L).The wet cake (4.28 kg) was dried under vacuum at 40±5° C. for 118 h toafford 3390 g of Compound A form I.

The X-ray Powder Diffraction study was performed on different lots ofCompound A morphic Form I generated by the process described above. XRPDafter micronization confirms Form 1.

The data for Form I is provided in Table 11 below and the diffractogramsof Form I are provided as FIG. 1.

TABLE 11 2θ d value Intensity Intensity (angle) (Å) (counts) % (%)3.0288 29.17117 1925.62 15.89 3.4596 25.5397 832.08 4.58 3.6702 24.07429707.65 3.89 4.0027 22.07529 410.45 6.78 4.4466 19.87232 432.4 2.384.5794 19.29632 429.89 4.73 5.2533 16.82257 320.41 5.29 5.8566 15.09082335.71 1.85 6.05 14.60887 224.56 9.89 6.8068 12.98624 287.97 3.17 7.215212.25213 293.93 4.04 7.6426 11.56781 239.85 2.64 8.2256 10.74918 1637.2713.51 8.8542 9.98745 309.91 3.41 9.115 9.70221 244.6 2.02 9.576 9.23622255.43 2.11 10.5373 8.39569 9763.54 100 11.1868 7.9096 2398.13 24.5613.0814 6.76802 164.19 3.36 13.9013 6.37063 197.28 1.52 14.3022 6.19296290.11 2.23 14.7284 6.01469 94.1 0.96 15.7399 5.63037 1305.28 16.7116.4002 5.40513 804.24 10.3 16.732 5.2987 173.26 2.22 17.3055 5.12435145.15 2.97 17.6872 5.01461 1400.39 17.93 18.3399 4.83761 1233.01 9.4718.6986 4.7456 9825.6 100 18.9598 4.6808 572.69 3.5 19.3018 4.59864278.53 1.7 19.6643 4.51468 97.55 0.4 20.0939 4.41912 64.71 2.63 21.06044.21845 333.65 2.72 22.2097 4.00268 833.43 8.48 22.6128 3.93224 1304.9510.62 22.8964 3.88417 3375.42 34.35 23.066 3.856 976.63 5.96 23.57423.77401 3115.33 38.05 23.8662 3.72849 571.62 4.65 24.1 3.69284 572.346.99 24.5243 3.62991 1097.27 6.7 24.6502 3.61166 1580.95 16.09 25.49933.49329 225.6 2.76 26.4933 3.36443 506.03 5.15 26.7528 3.33239 244.511.99 27.1244 3.28756 130.69 1.06 27.4354 3.251 546.35 4.45 27.83823.20487 213.44 2.17 28.5208 3.12971 158.82 1.29 28.9064 3.08883 436.592.67 29.1352 3.06509 710.53 5.79 29.5077 3.02724 416.16 4.24 30.02672.97608 1470.29 17.96 30.3658 2.94361 260.89 1.59 30.6326 2.91858 132.130.54 31.316 2.85644 177.78 1.45 31.6013 2.83129 397.61 5.67 31.92372.80343 514.26 4.19 32.2125 2.77895 1293.04 18.42 32.8721 2.72469 434.372.65 33.3755 2.68474 295.36 2.4 33.8232 2.65022 358.99 3.65 34.83642.57542 140.57 1.72 35.1838 2.55079 739.55 7.53 35.7301 2.51303 98.131.2 36.0084 2.49424 110.57 1.35 36.4676 2.46389 316.07 2.57 37.27472.41237 199.99 4.07 38.3543 2.34691 34.08 0.42 39.1941 2.29854 63.88 1.339.9663 2.25589 211.73 1.29 40.6489 2.21957 96.61 0.59 41.194 2.19145167.45 1.36 42.0276 2.14989 47.01 0.57 42.4477 2.12958 290.42 1.7742.8091 2.11244 200.71 1.63 43.6289 2.07463 171.28 2.09

Form I was found to have a melting onset around 321° C., followed bydecomposition upon melting by DSC (FIG. 2).

Example 7 Preparation of Compound A Form I: Conversion of Compound ASolvate to Form I

A 50 L, jacketed, glass vessel purged with N₂ was charged with CompoundA MIBK solvate (2.72 kg) from Example 5 above and ethanol (13.6 L). Theagitator was started and the batch temperature was adjusted from 16.8°C. to 79.4° C. over 1.3 h. The batch was held at 79.5° C. for 2 h andsampled for XRPD analysis. XRPD indicated Form I, and the batch wascooled to 24.9° C. over 1 h and 10 min. An 18″ Nutsche filter equippedwith tight-weave (0.67 CFM) polypropylene cloth was set up and the batchwas filtered. The filtration took 4 min. Ethanol (2.8 L) was charged tothe vessel and transferred to the filter cake. The cake was manuallyresuspended and the liquors were pulled through with vacuum. Ethanol(2.80 L) was charged to the filter cake and the cake was manuallyresuspended. The liquors were pulled through with vacuum and the cakewas conditioned with vacuum and nitrogen for 1 h. The filter cake wastransferred to drying pans and dried at 45° C. and 28″/Hg for one day togive an 89% yield (1.96 kg) of Compound A as a light yellow solid. HPLCanalysis indicated a purity of 99.6%. XRPD analysis is consistent withForm I. Micronization of 300 g of this material on a 2″ jet mill gave284 g (95% yield) of micronized Compound A. XRPD analysis confirmed thatmicronized Compound A remained Form I.

Compound A DMAC solvate can be converted, via the dihydrate and the MIBKsolvate, to Form I as described in Example 7. Alternatively, the DMACsolvate was converted directly to Form I in 75% yield (yield calculatedfrom Intermediate 8) by heating it with 8 volumes of ethanol to 80° C.for 2 hours followed by cooling to room temperature and filtering. Inanother reaction, a sample of Compound A that was a mixture of the DMACsolvate and dihydrate was converted to Form I in 69% yield by heating itwith 8 volumes of MIBK to 80° C. followed by cooling to roomtemperature.

Modeling of Interaction Between Compound a and Thyroid Hormone Receptor

Crystal structures were obtained from the RCSB protein data bank (IDnumbers: 1N46, 1NQ0, 1NQ1, 1NQ2 and 1NUO). The protein co-crystalstructures were aligned using MacPymol for Mac OS X (Copyright 2006DeLano Scientific LLC.; now a product of Schrodinger Inc.) MacPymol wasalso used for all analysis of the ligand-protein interactions and torender the FIGS. 3-9. These figures indicate that, overall, Compound Ais better able to accommodate the structural variations in the THRβmutants. For example, in mutant Arg316His, Arg316 is mutated to His andArg320 is slightly shifted away from ligand. As a result, the specificinteraction between Arg320 and T3 is less optimal in Arg316His mutant.In comparison, the large negative polarizable heterocycle in Compound Aforms favorable interactions that are not disrupted by Arg316Hismutation. In other words, Compound A, having a larger, more polarizableheterocycle, maintains favorable interactions with Arg320 and mutatedHis316. See, e.g., FIGS. 8 and 9. Results are similar for othermutations.

The table below lists the biochemical properties of certain TRβ mutants.Other mutants and their properties can be found in e.g., M. Adams etal., J Clin Invest. 1994; 94(2): 506-515, B. R. Huber et al., MolEndocrinol, 2003, 17(4):643-652; and B. R. Huber et al., Mol Endocrinol,2003, 17(1):107-116, the contents of each of which are herebyincorporated by reference in their entireties.

Trans- TRβ % T3 binding Activation Clinical WT 100    1X NormalAla234Thr High in solution,   .1X low in presence of (normal at thyroidresponse high T3) element DNA Arg243Gln High in solution, <.1X severedecrease in (normal at presence of thyroid very high response elementT3) DNA Ala317Thr 13 Normal at General resistance to 10XT3 thyroidhormone Arg316His .9 Normal at General resistance to high T3 thyroidhormone

Equivalents

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A morphic form of2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) (Form I) characterized by an X-ray powder diffractionpattern including peaks at about 10.5, 18.7, 22.9, 23.6, and 24.7degrees 2θ.
 2. The morphic form of claim 1 characterized by an X-raypowder diffraction pattern further including peaks at about 8.2, 11.2,15.7 16.4, 17.7, 30.0, and 32.2 degrees 2θ.
 3. The morphic form of claim1 characterized by an X-ray powder diffraction pattern substantiallysimilar to that set forth in FIG.
 1. 4. The morphic form of claim 1,wherein the morphic form has a purity of 95% or greater.
 5. Apharmaceutical composition comprising a morphic form of claim 1 and apharmaceutically acceptable carrier.
 6. A synthetic process comprising:(a) contacting R¹MgX or R¹Li with a compound of Formula (I):

 to form a compound of Formula (II):

 in which R¹ is isopropyl or isopropenyl, X is halo and R² is H or anamine protecting group; and (b) converting the compound of Formula (II)to a compound of Formula (III):

 in the presence of a base when R¹ is isopropenyl or in the presence ofan oxidizing agent when R¹ is isopropyl.
 7. The process of claim 6,further comprising: (c), when present, removing the amine protectinggroup R² of the compound of Formula (III) to form6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one.
 8. Theprocess of claim 6, wherein step (a) is performed by contacting R¹MgXwith the compound of Formula (I), in which R¹ is isopropenyl and X isBr; or step (a) is performed by contacting R¹MgX with the compound ofFormula (I), in which R¹ is isopropyl and X is Cl.
 9. The process ofclaim 6, wherein the oxidizing agent in step (b) is bromine and step (b)is performed in the presence of an acid.
 10. The process of claim 6,wherein R² is acetyl or benzoyl.
 11. The process of claim 7, furthercomprising: (d) converting6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one to2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile(“Compound A”) by contacting6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one withethyl (2-cyanoacetyl)carbamate and a metal nitrite followed by treatmentwith potassium acetate in DMAC.
 12. A process of preparing the preparingthe morphic form of claim 1, the process comprising: heating a mixturecomprising a solvent and Compound A at a first temperature, and coolingthe mixture to a second temperature that is lower than the firsttemperature so as to obtain the morphic form of claim 1, wherein thesolvent is selected from ethanol, isopropanol, methyl isobutyl ketoneand a combination thereof.
 13. The process of claim 12, wherein thefirst temperature is between about 60° C. and about 80° C.
 14. Theprocess of claim 12, wherein the second temperature is between about 0°C. and about 60° C.
 15. The process of claim 12, wherein the solvent isethanol or methyl isobutyl ketone.
 16. A process of preparing a morphicform of claim 1, the process comprising: heating a mixture comprising asolvent and Compound A at a first temperature, wherein the solvent isselected from ethanol, isopropanol, methyl isobutyl ketone and acombination thereof; cooling the mixture to a second temperature that islower than the first temperature; filtering the mixture to obtain afilter cake; rinsing the filter cake with an organic solvent to obtain arinsed filter cake; and drying the rinsed filter cake to obtain themorphic form of claim
 1. 17. A process of preparing a morphic form ofclaim 1, the process comprising: heating a mixture comprising ethanoland Compound A at a first temperature; cooling the mixture to a secondtemperature that is lower than the first temperature but greater thanabout 40° C.; filtering the mixture at a temperature not lower thanabout 40° C. to obtain a filter cake; rinsing the filter cake at atemperature not lower than about 40° C. with ethanol to obtain a rinsedfilter cake; and drying the rinsed filter cake at a temperature notlower than about 40° C. to obtain the morphic form of claim 1.